Xylose utilization in recombinant Zymomonas

ABSTRACT

Zymomonas  expressing xylose isomerase from  A. missouriensis  was found to have improved xylose utilization, growth, and ethanol production when grown in media containing xylose. Xylose isomerases related to that of  A. missouriensis  were identified structurally through molecular phylogenetic and Profile Hidden Markov Model analyses, providing xylose isomerases that may be used to improve xylose utilization.

This application claims the benefit of U.S. Provisional Application 61/359,463, filed Jun. 29, 2010 and is incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with United States government support under Contract Nos. DE-FC36-07GO17056 awarded by the Department of Energy. The government has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to the fields of microbiology and genetic engineering. More specifically, xylose isomerases with high activity in Zymomonas were identified that provide for improved xylose utilization and ethanol production.

BACKGROUND OF THE INVENTION

Production of ethanol by microorganisms provides an alternative energy source to fossil fuels and is therefore an important area of current research. It is desirable that microorganisms producing ethanol, as well as other useful products, be capable of using xylose as a carbon source since xylose is the major pentose in hydrolyzed lignocellulosic biomass. Biomass can provide an abundantly available, low cost carbon substrate. Zymomonas mobilis and other bacterial ethanologens which do not naturally utilize xylose have been genetically engineered for xylose utilization by introduction of genes encoding 1) xylose isomerase, which catalyses the conversion of xylose to xylulose; 2) xylulokinase, which phosphorylates xylulose to form xylulose 5-phosphate; 3) transketolase; and 4) transaldolase. Typically the coding regions used were from E. coli genes.

There has been success in engineering Z. mobilis strains for xylose metabolism (U.S. Pat. No. 5,514,583, U.S. Pat. No. 5,712,133, U.S. Pat. No. 6,566,107, WO 95/28476, Feldmann et al. (1992) Appl. Microbiol. Biotechnol. 38: 354-361, Zhang et al. (1995) Science 267:240-243), as well as a Zymobacter palmae strain (Yanase et al. (2007) Appl. Environ. Mirobiol. 73:2592-2599). However, typically the engineered strains do not grow and produce ethanol as well on xylose as on glucose. Strains engineered for xylose utilization have been adapted by serial passage on xylose medium, resulting in strains with improved xylose utilization as described in U.S. Pat. No. 7,223,575 and U.S. Pat. No. 7,741,119. Disclosed in commonly owned and co-pending US Patent Application Publication US 20090246846A1 is engineering for improved xylose utilization by expression of E. coli xylose isomerase from a mutated, highly active Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter (Pgap).

There remains a need for strains of Zymomonas, and other bacterial ethanolagens, which have further improvement in xylose utilization.

SUMMARY OF THE INVENTION

The invention provides recombinant xylose-utilizing Zymomonas or Zymobacter cells that express a highly active xylose isomerase providing improved xylose utilization and ethanol production.

Accordingly, the invention provides a recombinant bacterial strain selected from the group consisting of Zymomonas and Zymobacter comprising a heterologous nucleic acid molecule encoding a polypeptide having xylose isomerase activity wherein the polypeptide is a Group I xylose isomerase and is included in the class of enzymes identified by EC 5.3.1.5, and wherein the strain utilizes xylose as a carbon source.

Xylose isomerase enzymes useful in the invention are those that have an E-value score of 1E-15 or less when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, and 142; the query being carried out using the hmmsearch algorithm wherein the Z parameter is set to 1 billion, or those that have at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix; or those that have the following conserved amino acids, or 90% of the following conserved amino acids, when compared with the reference amino acid sequence of SEQ ID NO:66:

a) leucine at position 226,

b) methionine at position 223,

c) isoleucine at position 191,

d) threonine, serine, or valine at position 195,

e) methionine, threonine or guanine at position 88,

f) histidine at position 290,

g) glutamic acid or aspartic acid at position 221,

h) phenylalanine, valine, or leucine at position 242,

i) histidine at position 243,

j) leucine, phenylalanine, or methionine at position 193,

k) glutamine at position 256,

l) glycine at position 213,

m) proline, tyrosine, alanine, or serine at position 288, and

n) glutamine at position 249.

In another embodiment the invention provides a process for improving xylose utilization in a recombinant bacterial cell comprising:

-   -   a) providing a recombinant bacterial strain selected from the         group consisting of Zymomonas and Zymobacter comprising a xylose         utilization pathway comprising a xylose isomerase not belonging         to Group I; and     -   b) introducing a heterologous nucleic acid molecule encoding a         polypeptide having xylose isomerase activity wherein the         polypeptide is a Group I xylose isomerase.         Alternatively the invention provides a method for the production         of ethanol comprising:     -   a) providing the recombinant bacterial strain of the invention;         and     -   b) contacting the strain of (a) with xylose under conditions         whereby the strain produces ethanol.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

FIG. 1 shows a diagram of the ethanol fermentation pathway in Zymomonas engineered for xylose utilization.

FIG. 2 is a diagram of a phylogenetic tree of xylose isomerases showing Group I and Group II branches.

FIG. 3 is a diagram of a phylogenetic tree of Group I xylose isomerases.

FIG. 4 is a graph of growth curves for a control ZW641 strain and strains of ZW641 transformed with a gene having a codon-optimized coding region for expression of xylose isomerase from A. missourinesis (355-1, 355-2), L. brevis (356-1, 356-2), or E. coli (357-1, 357-2).

FIG. 5 is a graph of xylose isomerase activities in a control ZW641 strain and strains of ZW641 transformed with a gene having a codon-optimized coding region for expression of xylose isomerase from A. missourinesis (355-1), L. brevis (356-2), or E. coli (357-2) measured by the Cysteine-Carboazole method.

FIG. 6 is a graph of growth curves for a control ZW641 strain and strains of ZW641 transformed with a gene having a codon-optimized coding region for expression of xylose isomerase from A. missourinesis (AMxylA), E. coli (ECxylA), Geodermatophilus obscurus (GOxylA), Mycobacterium smegmatis (MSxylA), Salinispora arenicola (SAxylA), or Xylanimonas cellulosilytica (XCxylA).

FIG. 7 is a graph of xylose isomerase activities in a control ZW641 strain and strains of ZW641 transformed with a gene having a codon-optimized coding region for expression of xylose isomerase from A. missourinesis (AMxylA), E. coli (ECxylA), Geodermatophilus obscurus (GOxylA), Mycobacterium smegmatis (MSxylA), Salinispora arenicola (SAxylA), or Xylanimonas cellulosilytica (XCxylA) measured by the Cysteine-Carboazole method.

Table 1 is a table of the Profile HMM for xylose isomerase Group I proteins. Table 1 is submitted herewith electronically and is incorporated herein by reference.

Table 2 is a table of the E-value scores for XI proteins, each identified by a SEQ ID NO, that were queried using the Group I profile HMM. Table 2 is submitted herewith electronically and is incorporated herein by reference

The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions which form a part of this application.

The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

TABLE 3 SEQ ID numbers of Coding Regions and Proteins for Group I xylose isomerases. Uniprot accession number (AC) given for proteins that are seeds and NCBI GI number for given those that are not seeds. SEQ ID SEQ ID NO: NO: Nucleic Amino Organism GI or AC # acid acid Clavibacter michiganensis B0RIF1 1 2 Arthrobacter chlorophenolicus 220912923 3 4 Actinosynnema mirum 226865307 5 6 Kribbella flavida 227382478 7 8 Mycobacterium smegmatis 118469437 9 10 Arthrobacter sp. 60615686 11 12 Actinomyces urogenitalis 227497116 13 14 Streptomyces ambofaciens 126348424 15 16 Salinispora arenicola 159039501 17 18 Streptomyces sp. 38141596 19 20 Meiothermus silvanus 227989553 21 22 Actinoplanes sp. P10654 23 24 Mobiluncus curtisii 227493823 25 26 Herpetosiphon aurantiacus 159898286 27 28 Acidothermus cellulolyticus 117929271 29 30 Streptomyces coelicolor Q9L0B8 31 32 Streptomyces avermitilis Q93HF3 33 34 Nocardiopsis dassonvillei 229207664 35 36 Nakamurella multipartita 229221673 37 38 Xylanimonas cellulosilytica 227427650 39 40 Clavibacter michiganensis A5CPC1 41 42 Salinispora tropica 145596104 43 44 Streptomyces sp. 197764953 45 46 Streptomyces pristinaespiralis 197776540 47 48 Roseiflexus sp. 148656997 49 50 Meiothermus ruber 227992647 51 52 Arthrobacter sp. P12070 53 54 Thermobaculum terrenum 227374836 55 56 Janibacter sp. 84495191 57 58 Brachybacterium faecium 237671435 59 60 Beutenbergia cavernae 229821786 61 62 Geodermatophilus obscurus 227404617 63 64 Actinoplanes missouriensis P12851 65 66 Streptomyces violaceusniger P09033 67 68 Actinomyces odontolyticus 154508186 69 70 Mobiluncus mulieris 227875705 71 72 Cellulomonas flavigena 229243977 73 74 Saccharomonospora viridis 229886404 75 76 Streptomyces lividans Q9RFM4 77 78 Frankia sp. 158316430 79 80 Streptosporangium roseum 229851079 81 82 Nocardioides sp. 119716602 83 84 Kribbella flavida 227381155 85 86 Roseiflexus castenholzii 156742580 87 88 Arthrobacter aurescens 119964059 89 90 Leifsonia xyli 50954171 91 92 Jonesia denitrificans 227383768 93 94 Streptomyces olivaceoviridis Q93RJ9 95 96 Stackebrandtia nassauensis 229862570 97 98 Thermus thermophilus P26997 99 100 Acidobacteria bacterium 94967932 101 102 Catenulispora acidiphila 229246901 103 104 Streptomyces corchorusii Q9S3Z4 105 106 Streptomyces thermocyaneoviolaceus Q9L558 107 108 marine actinobacterium 88856315 109 110 Micromonospora sp. 237882534 111 112 Thermobifida fusca 72162004 113 114 Herpetosiphon aurantiacus 159897776 115 116 Streptomyces griseus 182434863 117 118 Mycobacterium vanbaalenii 120406242 119 120 Streptomyces diastaticus P50910 121 122 Deinococcus geothermalis 94972159 123 124 Arthrobacter sp. A0JXN9 125 126 Streptomyces rubiginosus P24300 127 128 Streptomyces murinus P37031 129 130 Thermus caldophilus 4930285 * 131 Thermus caldophilus P56681 * 132 Arthrobacter sp. 231103 * 133 Actinoplanes missouriensis 443486 * 134 Streptomyces olivochromogenes P15587 * 135 Streptomyces olivochromogenes 157879319 * 136 Streptomyces rochei P22857 * 137 Streptomyces olivochromogenes 157881044 * 138 Streptomyces diastaticus 7766813 * 139 Actinoplanes missouriensis 349936 * 140 Arthrobacter sp. 2914276 * 141 Streptomyces albus P24299 * 142 Actinoplanes missouriensis 443303 * 143 Streptomyces diastaticus 9256915 * 144 Actinoplanes missouriensis 443526 * 145 Streptomyces rubiginosus 21730246 * 146 Actinoplanes missouriensis 443568 * 147 * Sequence not readily available

TABLE 4 SEQ ID numbers of Proteins for Group II seed xylose isomerases. Uniprot accession number (AC) given for the seed proteins. SEQ ID NO: Organism AC # Amino acid Salmonella enterica B4T952 148 Klebsiella pneumoniae P29442 149 Sinorhizobium meliloti Q92LW9 150 Escherichia coli Q7A9X4 151 Salmonella enterica Q5PLM6 152 Xanthomonas campestris Q3BMF2 153 Pectobacterium atrosepticum Q6DB05 154 Rhodopirellula baltica Q7UVG2 155 Xanthomonas axonopodis Q8PEW5 156 Xanthomonas oryzae Q5GUF2 157 Pediococcus pentosaceus Q03HN1 158 Brucella suis Q8G204 159 Escherichia coli Q0TBN7 160 Bifidobacterium longum Q8G3Q1 161 Brucella canis A9M9H3 162 Burkholderia multivorans A9ARG7 163 Brucella ovis A5VPA1 164 Rhizobium etli B3Q0R5 165 Burkholderia xenovorans Q13RB8 166 Actinobacillus pleuropneumoniae A3N3K2 167 Burkholderia cenocepacia B4ENA5 168 Solibacter usitatus Q022S9 169 Brucella abortus B2SA37 170 Rhodobacter sphaeroides A4WVT8 171 Thermoanaerobacter sp. B0K1L3 172 Yersinia pseudotuberculosis Q1C0D3 173 Xanthomonas oryzae Q5GYQ7 174 Bifidobacterium longum B3DR33 175 Thermoanaerobacter pseudethanolicus P22842 176 Photobacterium profundum Q6LUY7 177 Escherichia coli B1LJC7 178 Agrobacterium tumefaciens Q8U7G6 179 Tetragenococcus halophilus O82845 180 Salmonella enterica B4TZ55 181 Yersinia pseudotuberculosis Q8Z9Z1 182 Yersinia pseudotuberculosis Q1CDB8 183 Rhodobacter sphaeroides A3PNM4 184 Brucella abortus Q2YMQ2 185 Salmonella enterica Q8ZL90 186 Bacteroides vulgatus A6L792 187 Xanthomonas axonopodis Q8PLL9 188 Salmonella enterica Q57IG0 189 Escherichia coli B7M3I8 190 Roseobacter denitrificans Q162B6 191 Bacteroides fragilis Q64U20 192 Enterobacter sakazakii A7MNI5 193 Brucella abortus Q57EI4 194 Geobacillus thermodenitrificans A4IP67 195 Bacteroides thetaiotaomicron Q8A9M2 196 Haemophilus influenzae A5UCZ3 197 Yersinia pseudotuberculosis B2K7D2 198 Xanthomonas campestris Q4UTU6 199 Haemophilus somnus B0UT19 200 Pseudoalteromonas atlantica Q15PG0 201 Escherichia fergusonii B7LTH9 202 Silicibacter sp. Q1GKQ4 203 Salmonella enterica B5R4P8 204 Bifidobacterium adolescentis A1A0H0 205 Staphylococcus xylosus P27157 206 Thermotoga maritima Q9X1Z5 207 Salmonella enterica A9MUV0 208 Pseudomonas syringae Q48J73 209 Shigella boydii Q31V53 210 Burkholderia ambifaria Q0B1U7 211 Bacillus amyloliquefaciens A7Z522 212 Haemophilus influenzae A5UIN7 213 Bacillus megaterium O08325 214 Arabidopsis thaliana Q9FKK7 215 Escherichia coli Q3YVV0 216 Bacteroides fragilis Q5LCV9 217 Pseudomonas fluorescens Q3KDW0 218 Escherichia coli B1X8I1 219 Bacillus subtilis P04788 220 Xanthomonas campestris Q4UNZ4 221 Pseudomonas syringae Q4ZSF5 222 Sinorhizobium medicae A6UD89 223 Ochrobactrum anthropi A6X4G3 224 Burkholderia thailandensis Q2SW40 225 Salmonella enterica B5EX72 226 Thermotoga sp. B1LB08 227 Bacillus cereus Q739D2 228 Salmonella enterica B4SWK9 229 Salmonella enterica Q7C637 230 Enterococcus faecalis Q7C3R3 231 Thermotoga neapolitana P45687 232 Escherichia coli B7MES1 233 Photorhabdus luminescens Q7N4P7 234 Enterobacter sp. A4W566 235 Burkholderia cenocepacia B1KB47 236 Bacillus licheniformis P77832 237 Geobacillus stearothermophilus P54273 238 Brucella abortus Q8YFX5 239 Rhizobium leguminosarum Q1MBL8 240 Yersinia enterocolitica A1JT10 241 Serratia proteamaculans A8G7W8 242 Yersinia pseudotuberculosis A7FP68 243 Escherichia coli B7NEL7 244 Yersinia pestis A9R5Q1 245 Fervidobacterium gondwanense Q6T6K9 246 Xanthomonas campestris Q8P3H1 247 Rhizobium leguminosarum B5ZQV6 248 Bradyrhizobium japonicum Q89VC7 249 Mesorhizobium sp. Q11EH9 250 Actinobacillus pleuropneumoniae B3H2X9 251 Yersinia pseudotuberculosis Q663Y3 252 Xanthomonas campestris Q8P9T9 253 Burkholderia cenocepacia A0KE56 254 Oceanobacillus iheyensis Q8ELU7 255 Brucella suis B0CKM9 256 Thermoanaerobacterium P29441 257 thermosaccharolyticum Burkholderia phymatum B2JFE9 258 Yersinia pseudotuberculosis B1JH40 259 Bacillus sp. P54272 260 Lactococcus lactis Q02Y75 261 Novosphingobium aromaticivorans Q2GAB9 262 Lactobacillus brevis P29443 263 Mesorhizobium loti Q98CR8 264 Escherichia coli A8A623 265 Burkholderia cenocepacia Q1BG90 266 Thermoanaerobacterium thermosulfurigenes P19148 267 Thermotoga petrophila A5ILR5 268 Lactobacillus pentosus P21938 269 Lactococcus lactis Q9CFG7 270 Ruminococcus flavefaciens Q9S306 271 Burkholderia phytofirmans B2T929 272 Salmonella enterica B5FLD6 273 Lactobacillus brevis Q03TX3 274 Burkholderia ambifaria B1Z405 275 Salmonella enterica B5RGL6 276 Bacillus halodurans Q9K993 277 Bacillus clausii Q5WKJ3 278 Marinomonas sp. A6VWH1 279 Yersinia pseudotuberculosis A4TS63 280 Actinobacillus pleuropneumoniae B0BTI9 281 Silicibacter pomeroyi Q5LV46 282 Xanthomonas oryzae Q2NXR2 283 Thermoanaerobacterium saccharolyticum P30435 284 Escherichia coli B6I3D6 285 Escherichia coli B5YVL8 286 Escherichia coli B7NP65 287 Escherichia coli B2U560 288 Escherichia coli B1IZM7 289 Rhizobium etli Q2K433 290 Escherichia coli P00944 291 Hordeum vulgare Q40082 292 Dinoroseobacter shibae A8LP53 293 Rhodobacter sphaeroides Q3IYM4 294 Actinobacillus succinogenes A6VLM8 295 Bacillus pumilus A8FE33 296 Escherichia coli Q8FCE3 297 Pseudomonas syringae Q880Z4 298 Burkholderia vietnamiensis A4JSU5 299 Escherichia coli A7ZTB2 300 Haemophilus influenzae P44398 301 Haemophilus influenzae Q4QLI2 302 Listeria welshimeri A0AF79 303 Thermoanaerobacter yonseiensis Q9KGU2 304 Geobacillus kaustophilus Q5KYS6 305 Mannheimia succiniciproducens Q65PY0 306

SEQ ID NO:307 is the nucleotide sequence of the LoxPw-aadA-LoxPw DNA fragment PCR product.

SEQ ID NO:308 is the coding region for the Actinoplanes missourinesis xylose isomerase that was codon optimized for Zymomonas.

SEQ ID NO:309 is the coding region for the Lactobacillus brevis xylose isomerase that was codon optimized for Zymomonas.

SEQ ID NO:310 is the coding region for the E. coli xylose isomerase that was codon optimized for Zymomonas.

SEQ ID NO:311 is the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene promoter from Z. mobilis (ZmPgap).

SEQ ID NO:312 is the nucleotide sequence of the terminator from the Z. mobilis L-ribulose 5 phosphate 4-epimerase gene.

SEQ ID NO:313 is the nucleotide sequence of the pARA354 plasmid.

SEQ ID NO:314-327, 329, 330, and 332-334 are nucleotide sequences of PCR and sequencing primers.

SEQ ID NO:328 is the nucleotide sequence of the LDH-L DNA fragment PCR product.

SEQ ID NO:331 is the nucleotide sequence of the LDH-R DNA fragment PCR product.

SEQ ID NO:335 is the nucleotide sequence of the codon optimized coding region for Geodermatophilus obscurus xylose isomerase.

SEQ ID NO:336 is the nucleotide sequence of the codon optimized coding region for Mycobacterium smegmatis xylose isomerase.

SEQ ID NO:337 is the nucleotide sequence of the codon optimized coding region for Salinispora arenicola xylose isomerase.

SEQ ID NO:338 is the nucleotide sequence of the codon optimized coding region for Xylanimonas cellulosilytica xylose isomerase.

SEQ ID NO:339 is the nucleotide sequence of the promoter P_(gapS) used in chimeric gene constructions.

SEQ ID NOs:340-345 are nucleotide sequences of PCR and sequencing primers.

DETAILED DESCRIPTION

Disclosed herein are xylose isomerase enzymes that are highly active in Zymomonas, and may be used to increase xylose utilization and ethanol production. Ethanol is an important compound for use in replacing fossil fuels.

The following definitions may be used for the interpretation of the claims and specification:

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.

As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

The term “carbon substrate” or “fermentable carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides.

“Gene” refers to a nucleic acid fragment that expresses a specific protein or functional RNA molecule, which may optionally include regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” or “wild type gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.

The term “genetic construct” refers to a nucleic acid fragment that encodes for expression of one or more specific proteins or functional RNA molecules. In the gene construct the gene may be native, chimeric, or foreign in nature. Typically a genetic construct will comprise a “coding sequence”. A “coding sequence” refers to a DNA sequence that encodes a specific amino acid sequence.

“Promoter” or “Initiation control regions” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.

The term “expression”, as used herein, refers to the transcription and stable accumulation of coding (mRNA) or functional RNA derived from a gene. Expression may also refer to translation of mRNA into a polypeptide. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.

The term “transformation” as used herein, refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. The transferred nucleic acid may be in the form of a plasmid maintained in the host cell, or some transferred nucleic acid may be integrated into the genome of the host cell. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid” and “vector” as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “selectable marker” means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest.

As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.

The term “fermentable sugar” refers to oligosaccharides and monosaccharides that can be used as a carbon source by a microorganism in a fermentation process.

The term “lignocellulosic” refers to a composition comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose.

The term “cellulosic” refers to a composition comprising cellulose and additional components, including hemicellulose.

The term “saccharification” refers to the production of fermentable sugars from polysaccharides.

The term “pretreated biomass” means biomass that has been subjected to thermal, physical and/or chemical pretreatment to increase accessibility of polysaccharides in the biomass prior to saccharification.

“Biomass” refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.

“Biomass hydrolysate” refers to the product resulting from saccharification of biomass. The biomass may also be pretreated or pre-processed prior to saccharification.

The term “xylose isomerase” refers to an enzyme that catalyzes the interconversion of D-xylose and D-xylulose. Xylose isomerases (XI) belong to the group of enzymes classified as EC 5.3.1.5.

The term “E-value”, as known in the art of bioinformatics, is “Expect-value” which provides the probability that a match will occur by chance. It provides the statistical significance of the match to a sequence. The lower the E-value, the more significant the hit.

The term “Group I xylose isomerase” refers herein to a xylose isomerase protein that belongs to Group I as defined by at least one of the following criteria: a) it falls within a 50% threshold sequence identity grouping that includes the A. missouriensis XI that is prepared using molecular phylogenetic bioinformatics analysis as in Example 4; b) it substantially fits the amino acids for Group I in the specificity determining positions (SDP) identified using GroupSim analysis of the Group I and Group II XI sets determined from molecular phylogenetic analysis that are given in Table 6 in Example 4; and/or c) it has an E-value of 1E-15 or less when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, and 142; where the query is carried out using the hmmsearch algorithm with the Z parameter is set to 1 billion, as in Example 4. It is understood that although “Group 1” xylose isomerases are known and defined in the literature that the definition provided herein is more precise than the literature definition and is the definition that informs the following discussion. Thus, “Group I” as used herein will refer to Applicants' definition whereas “Group II” will refer to the definitions as commonly understood in the art.

The term “heterologous” means not naturally found in the location of interest. For example, a heterologous gene refers to a gene that is not naturally found in the host organism, but that is introduced into the host organism by gene transferIn addition, a heterologous nucleic acid molecule that is present in a chimeric gene is a nucleic acid molecule that is not naturally found associated with the other segments of the chimeric gene, such as the nucleic acid molecules having the coding region and promoter segments not naturally being associated with each other.

As used herein, an “isolated nucleic acid molecule” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe. The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.

The terms “homology” and “homologous” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.

Moreover, the skilled artisan recognizes that homologous nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under moderately stringent conditions (e.g., 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein.

The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).

Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

Additionally the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100% may be useful in describing the present invention, such as 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W. Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. et al., In Current Protocols in Molecular Biology, published by Greene Publishing and Wiley-Interscience, 1987.

The present invention relates to engineered strains of xylose-utilizing Zymomonas or Zymobacter that have improved xylose utilization when fermented in xylose containing media. A challenge for improving ethanol production by fermentation of a biocatalyst in media that includes biomass hydrolysate, produced typically by pretreatment and saccharification of biomass, is obtaining optimal utilization of xylose. Xylose is one of the predominant pentose sugars in hydrolyzed lignocellulosic materials, the other being arabinose. Applicants have identified a group of xylose isomerases that when expressed in xylose-utilizing Zymomonas strains provide for increased efficiency in xylose utilization and higher ethanol yields, when fermentation is in xylose containing media.

Discovery of Highly Effective Xylose Isomerases

Xylose isomerase (XI), which catalyzes the conversion of xylose to xylulose, is one of four enzymes that when expressed in a bacterial cell provide the ability to utilize xylose as a carbon source. XI along with xylulokinase, transketolase, and transaldolase provide a pathway from xylose that produces fructose-6-P and glyceraldehyde-3-P that feed into ethanol biosynthesis as shown in FIG. 1. As the first enzyme of the xylose utilization pathway, xylose isomerase activity is particularly important in providing the ability to ultimately convert xylose to ethanol. Applicants have identified xylose isomerases that have higher activity when expressed in the ethanol producing bacteria, Zymomonas, and support enhanced xylose utilization and ethanol production, as compared to the typically used E. coli XI.

The xylose isomerase from E. coli has been used in engineering Zymomonas for xylose utilization. Xylose isomerases are classified in two groups based on their size, amino acid sequence similarity, and divalent cation preference (Park and Batt (2004) Applied and Environmental Microbiology 70:4318-4325). The E. coli XI belongs to Group II.

Applicants have discovered that XIs belonging to Group I (as defined herein) provide enhanced properties for xylose utilization and ethanol production in xylose-utilizing Zymomonas. The Actinoplanes missouriensis XI was found herein to be a Group I XI as described below. When expressed in Zymomonas using a codon-optimized coding sequence (SEQ ID NO:308), the XI from Actinoplanes missouriensis had a xylose isomerase specific activity that was higher than specific activities of similarly expressed E. coli XI (using a codon-optimized coding sequence: SEQ ID NO:310), or another Group II XI, that of Lactobacillus brevis (codon-optimized coding sequence: SEQ ID NO:309). The codon-optimized coding sequences for the A. missouriensis, E. coli, and L. brevis XIs were each expressed in Zymomonas engineered with all four enzymes for xylose utilization, including the E. coli xylose isomerase expressed from a non-optimized coding sequence. Strains expressing the A. missouriensis XI grew better in xylose containing medium, utilized more xylose, and produced more ethanol than strains expressing the XI from either E. coli or L. brevis.

Zymomonas strains expressing additional XIs that were identified as belonging to Group I, as described below, were also found to grow better, utilize more xylose and produce more ethanol in xylose containing medium than strains expressing the Group II XI from E. coli or L. brevis. These were strains containing the XIs from Geodermatophilus obscurus (SEQ ID NO:64), Mycobacterium smegmatis (SEQ ID NO:10), Salinispora arenicola (SEQ ID NO:18), and Xylanimonas cellulosilytica (SEQ ID NO:40).

The growth enhancement, xylose utilization improvement, and ethanol production improvement each may vary in extent in a Group I XI expressing strain as compared to a Group II XI expressing strain. Differences may be based on factors such as specific XI encoding gene expression properties, culturing conditions such as carbon source composition in the medium including amount of xylose and other carbon sources, and strain characteristics such as additional genetic modifications involved in xylose utilization and/or ethanol production.

Group I Xylose Isomerases

Any XI belonging to Group I may be used in the present strains to improve xylose utilization. The Group I XIs have been distinguished from Group II XIs by their length. Group I XIs were found typically to be about 380 to 390 amino acids in length while Group II XIs are typically about 440 to 460 amino acids in length. Among Group I XIs there is amino acid identity of at least about 50%, while Group II XIs have only 20-30% amino acid identity with Group I XIs. Thus XIs can be readily classified as belonging to Group I or Group II using these structural criteria.

XIs identified in Park and Batt (supra) as belonging to Group I are those from Streptomyces, Actinoplanes, Thermus, and Arthrobacter while XIs identified as belonging to Group II are those from Klebsiella, Escherichia, lactobacillus, Lactococcus, Clostridium, Bacillus, Staphylococcus, and Thermoanaerobacter.

Bioinformatics analysis was used to more fully characterize Group I as opposed to Group II XIs to identify those XIs that may be used in the present strains. Members of Group I xylose isomerases were identified using molecular phylogenetic analysis of XI amino acid sequences. The molecular phylogenetic analysis was performed on 444 XI sequences collected from a public database using multiple query sequence BLAST analysis (blastall) using 180 XI seed sequences with functional annotations retrieved from the SWISSPROT database: SEQ ID NOs:2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, 142 and 148-306.

The resulting phylogenetic tree shown in FIG. 2 places the A. missouriensis XI in one phylogenetic grouping, called Group I, and the E. coli XI in a separate grouping, called Group II. Group I and Group II are labeled to coincide with the groupings of Park and Bratt (supra). The Lactobacillus brevis XI, tested herein in Example 3 and shown to have comparable effects as the XI from E. coli, is also in Group II. Twenty-one of the seed sequences (SEQ ID NOs:2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, and 142) were found to belong to Group I XIs. The identified XI sequences belonging to Group I form a 50% threshold identity cluster, as described in Example 4. XI sequences belonging to Group II form a separate 50% threshold identity cluster. Each identified Group I XI is encoded by the genome of, and therefore is endogenous to, one of the following microorganisms: Actinoplanes, Arthrobacter, Streptomyces, Thermus, Thermobaculum, Herpetosiphon, Acidobacteria, Roseiflexus, Meiothermus, Deinococcus, Meiothermus, Stackebrandtia, Kribbella, Xylanimonas, Nocardiopsis, Catenulispora, Streptosporangium, Geodermatophilus, Actinosynnema, Saccharomonospora, Acidothermus, Tthermobifida, Nocardiodes, Janibacter, Mycobacterium, Leifsonia, Clavibacter, Micromonospora, Salinispora, Cellulomonas, Jonesia, Nakamurella, Actinomyces, Mobiluncus, Brachybacterium, Beutengergai, Frankia, and Actinobacterium.

Any XI that belongs to Group I XIs as determined by molecular phylogenetic analysis as described in Example 4 herein may be used in the present strains. The molecular phylogeny of the Group I XIs is shown in more detail in FIG. 3. The Group I XIs in FIG. 3 are listed in Table 3 as having SEQ ID NOs that are even numbers between 2 and 130 and 131-147. Coding regions for these proteins are listed in Table 3 as having SEQ ID NOs that are odd numbers between 1 and 129. Any other identified XI that can be identified as belonging to Group I using molecular phylogenetic analysis as described in Example 4 herein may be used in the present strains. Alternatively, XIs that may be used in the present strains may be identified as xylose isomerase proteins with amino acid sequences having at least about 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95%, or at least about 96%, 97%, 98%, or 99% sequence identity to any of the XI amino acid sequences of SEQ ID NOs that are even numbers between, and including, 2 and 130 and 131-147. In one embodiment XIs that may be used in the present strains may be identified as proteins with xylose isomerase activity and with amino acid sequences having at least about 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95%, or at least about 96%, 97%, 98%, or 99% sequence identity to any of the XI amino acid sequences of SEQ ID NOs: 24, 66, 134, 140, 143, 145, and 147. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Group I and Group II XIs were further characterized using GroupSim analysis as described in Example 4 herein. Through this analysis specific amino acid positions were determined to be specificity determining positions (SDP) for distinguishing the structures of Group I and Group II XI proteins. The locations of these SDP amino acids are given here at corresponding positions in the representative Group I protein P12851 from Actinoplanes missouriensis (SEQ ID NO:66) and in the representative Group II protein P19148 from Thermoanaerobacterium thermosulfurigenes (SEQ ID NO:267). The positions in the Group II protein are generally about 51 greater than in the Group I protein. The corresponding positions in other Group I and II proteins can readily be identified by one skilled in the art by sequence alignment and context. The SDP identifiers distinguishing Group I and Group II XIs with a score of 0.9 or greater (where a perfect score of 1 would indicate that all proteins within the group have the listed amino acid in the specified position and between groups the amino acid is always different) are the following amino acid (AA) positions in P12851 vs in P19148:

1) AA 226 is leucine; AA277 is histidine

2) AA223 is methionine; AA274 is leucine

3) AA191 is isoleucine; AA242 is glutamine

4) AA195 is threonine, serine, or valine; AA246 is aspartic acid

5) AA88 is methionine, threonine, or guanine; AA139 is arginine or tryptophan

6) AA290 is histidine; AA337 is asparagine or methionine

7) AA221 is glutamic acid or aspartic acid; AA 272 is alanine, threonine, or glycine

8) AA242 is phenylalanine, valine, or leucine; A293 is glycine, cysteine, or tryptophan

9) AA243 is histidine; AA294 is serine, asparagine, glycine, or leucine

10) AA193 is leucine, phenylalanine, or methionine; AA244 is aspartic acid

11) AA256 is glutamine; AA 308 is threonine, isoleucine, valine, leucine, methionine, tyrosine, or histidine

12) AA213 is glycine; AA264 is lysine, asparagine, serine, glutamic acid, alanine, leucine, arginine, or glutamine

13) AA288 is proline, tyrosine, alanine, or serine; AA335 is valine or glycine

14) AA249 is glutamine; AA301 is aspartic acid, histidine, asparagine, arginine, serine, or alanine

Using these amino acid position identifiers, a Group I XI can be readily identified by one skilled in the art. An XI having the Group I amino acid position identifiers substantially as listed above will be considered herein to be a Group I XI irrespective of length. Thus if substantially all of the positions indicated for Group II XIs are filled by the amino acids indicated for a Group I XI, the protein is considered to be a Group I XI regardless of length. For example, an XI having the Group I AA226 leucine identifier at the 277 position instead of histidine is a Group I XI if this pattern holds for the other amino acid identifiers. Substantially here means that there need not be a complete exact match at all positions, as indicated by the scores of 0.9 as opposed to a score of 1. A Group I XI has at least 90% of the amino acids at the SDPs matching the above list.

An additional bioinformatics analysis of Group I XIs was performed using the hmmsearch algorithm of the HMMER software package (Janelia Farm Research Campus, Ashburn, Va.). The Z parameter of the hmmsearch algorithm was set to 1 billion. The output of the HMMER analysis using a set of protein sequences is a Profile Hidden Markov Model (Profile HMM). The theory behind Profile HMMs as described in Durbin et al. (Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998) and Krogh et al. (1994 J. Mol. Biol. 235:1501-1531), both incorporated herein by reference, is characterization of a set of proteins based on the probability of each amino acid occurring at each position in the alignment of the proteins of the set.

The 21 seed sequences having known xylose isomerase activity and found to belong to Group I XIs in the molecular phylogenetic analysis described above were used as the set of proteins to prepare a profile HMM. These proteins have SEQ ID NOs:2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, and 142. All of the XIs that were identified by molecular phylogenetic analysis as belonging to Group I, which are listed in Table 3, match the profile HMM prepared from Group I seed sequences with E-value scores of less than or equal to 2.2e-181, with the Z parameter set to 1 billion. All of the XIs identified by molecular phylogenetic analysis as belonging to Group II match the same profile HMM with E-value scores of greater than or equal to 1.5e-07. Table 2 of the appendix lists the E-value scores for each XI SEQ ID NO. Thus the prepared profile HMM gives a structural characterization for functional Group I XIs and corroborates the molecular phylogenetic analysis. Therefore any XI protein that matches the profile HMM prepared using the 21 Group I XI seed sequences described above with an E-value of 1E-15 or less, where 1E-15 is between the highest score for a Group I XI and the lowest score for a Group II XI, may be used in the present strains. Lower E-value scores indicate better matches.

Additionally, the Group I XI sequences described herein or those recited in the art may be used to identify other homologs in nature. For example each of the XI encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1) methods of nucleic acid hybridization; 2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3) methods of library construction and screening by complementation.

As is known in the art, there may be variations in DNA sequences encoding an amino acid sequence due to the degeneracy of the genetic code. Codons may be optimized for expression of an amino acid sequence in a target host cell to provide for optimal encoded protein expression

Group I XI Expression

In the present strains, any of the Group I XIs described above may be expressed in a strain of Zymomonas or related ethanolagen, such as Z. mobilis or Zymobacter, along with genes encoding xylulokinase, transketolase, and transaldolase as described above, which then is capable of utilizing xylose as described below. Zymobacter palmae is an ethanol-producing bacterium that has been engineered for xylose utilization by expressing genes for xylose utilization as described below for Zymomonas, using Z. mobilis glyceraldehyde-3-phosphate dehydrogenase and enolase promoters (Yanase et al. Applied and Environmental Microbiology (2007) 73:2592-2599).

Coding regions that may be used to express Group I XIs include those listed in Table 1 as SEQ IDs with odd numbers from 1 through 129, other sequences encoding XI proteins listed in Table 1 as SEQ IDs with even numbers 2 through 130 and 131-147, as well as sequences identified in the art as encoding Group I XIs using bioinformatics or experimental methods described herein and those well known in the art.

For expression, a Group I XI coding region is constructed in a chimeric gene with operably linked promoter and typically a termination sequence. Alternatively the Group I XI coding region is constructed as part of an operon that is operably linked to a promoter and a termination sequence, and includes one or more additional coding regions. Promoters that may be used are promoters that are expressed in Zymomonas or Zymobacter cells such as the promoters of Z. mobilis glyceraldehyde-3-phosphate dehydrogenase (GAP promoter or P_(gap)), including mutant more highly active GAP promoters disclosed in US 20090246876, which is incorporated herein by reference, that may be called superGAP promoters or P_(gapS), and Z. mobilis enolase (ENO promoter) genes. Termination signals are also those that are expressed in the target cell.

A chimeric gene or operon for XI expression is typically constructed in or transferred to a vector for further manipulations. Vectors are well known in the art. Certain vectors are capable of replicating in a broad range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors: pRK437, pRK442, and pRK442(H) are available. These derivatives have proven to be valuable tools for genetic manipulation in gram-negative bacteria (Scott et al., Plasmid 50(1):74-79 (2003)).

Particularly useful for expression in Zymomonas are vectors that can replicate in both E. coli and Zymomonas, such as pZB188 which is described in U.S. Pat. No. 5,514,583. Vectors may include plasmids for autonomous replication in a cell, and plasmids for carrying constructs to be integrated into bacterial genomes. Plasmids for DNA integration may include transposons, regions of nucleic acid sequence homologous to the target bacterial genome, or other sequences supporting integration. An additional type of vector may be a transposome produced using, for example, a system that is commercially available from EPICENTRE®. It is well known how to choose an appropriate vector for the desired target host and the desired function.

Bacterial cells may be engineered by introducing a vector having a chimeric gene comprising a xylose isomerase coding region by well known methods, such as using freeze-thaw transformation, calcium-mediated transformation, electroporation, or conjugation. Any bacterial cell to be engineered for xylose utilization by expressing a xylose isomerase enzyme is a target host cell for transformation to engineer a strain as described herein. Particularly suitable host cells are Zymomonas and Zymobacter cells. The introduced chimeric gene may be maintained in the cell on a stably replicating plasmid, or integrated into the genome following introduction.

For engineering a strain with an integrated xylose isomerase chimeric gene or operon in the bacterial cell genome, methods may be used that are well known in the art such as homologous recombination, transposon insertion, or transposome insertion. In homologous recombination, DNA sequences flanking a target integration site are placed bounding a spectinomycin-resistance gene, or other selectable marker, and xylose isomerase chimeric gene leading to insertion of the selectable marker and the xylose isomerase chimeric gene into the target genomic site. In addition, the selectable marker may be bounded by site-specific recombination sites, so that after expression of the corresponding site-specific recombinase, the resistance gene is excised from the genome.

Engineering of Full Xylose Utilization Pathway

In addition to transforming with a chimeric gene or operon comprising a Group I XI coding region, the present strains are also engineered for expression of three other enzymes needed for xylose utilization: xylulokinase, which phosphorylates xylulose to form xylulose 5-phosphate, and transaldolase and transketolase, two enzymes of the pentose phosphate pathway which convert xylulose 5-phosphate to intermediates that couple pentose metabolism to the glycolytic Entner-Douderoff pathway permitting the metabolism of xylose to ethanol (see FIG. 1). Xylose utilizing Zymomonas strains are described in U.S. Pat. No. 5,514,583, U.S. Pat. No. 5,712,133, U.S. Pat. No. 6,566,107, WO 95/28476, Feldmann et al. ((1992) Appl Microbiol Biotechnol 38: 354-361), Zhang et al. ((1995) Science 267:240-243. These strains were transformed with coding sequences from E. coli genes, including a Group II xylose isomerase.

The Group I XI may be the sole XI expressed for xylose utilization-, or it may be expressed in addition to an expressed Group II XI such as that from E. coli. Thus a Group I XI may be introduced in a Zymomonas or Zymobacter strain that has a full xylose utilization pathway that includes a Group II xylose isomerase encoding gene and is capable of utilizing xylose. Alternatively, a Group I XI may be introduced in a Zymomonas or

Zymobacter strain that expresses xylulokinase, transaldolase, and transketolase, and only lacks xylose isomerase activity for utilizing xylose. With introduction of a Group I XI the strain is capable of utilizing xylose.

The additional three enzymes may be expressed from individual chimeric genes or from operons including more than one coding region as well known to one skilled in the art. DNA sequences encoding these enzymes may be obtained from any of numerous microorganisms that are able to metabolize xylose, such as enteric bacteria, and some yeasts and fungi. Sources for the coding regions include Xanthomonas, Klebsiella, Escherichia, Rhodobacter, Flavobacterium, Acetobacter, Gluconobacter, Rhizobium, Agrobacterium, Salmonella, Pseudomonads, and Zymomonas. Particularly useful are the coding regions of E. coli.

Endogenous genes may provide part of a xylose fermentation pathway, or may be altered by any known genetic manipulation technique to provide a protein with enzyme activity useful for xylose metabolism. For example, an endogenous transketolase may complement other introduced enzyme activities in creating a xylose utilization pathway.

Examples of xylose-utilizing strains that are known and may be used include CP4(pZB5) (U.S. Pat. No. 5,514,583), ATCC31821/pZB5 (U.S. Pat. No. 6,566,107), 8b (US 20030162271; Mohagheghi et al., (2004) Biotechnol. Lett. 25; 321-325), and ZW658 (ATTCC #PTA-7858; U.S. Pat. No. 7,741,119).

Zymomonas or Zymobacter strains that are additionally engineered to utilize other sugars that, like xylose, are not natural substrates, may also be used in the present process. An example is a strain of Z. mobilis engineered for arabinose utilization as described in U.S. Pat. No. 5,843,760, which is herein incorporated by reference. Strains may be modified in other additional ways to improve xylose utilization and ethanol production.

Fermentation Of Improved Xylose-Utilizing Strain

An engineered xylose-utilizing strain having a Group I xylose isomerase chimeric gene and genes or operons for expression of xylulokinase, transaldolase and transketolase may be used in fermentation to produce a product that is a natural product of the strain, or a product that the strain is engineered to produce. For example, Zymomonas mobilis and Zymobacter palmae are natural ethanolagens. As an example, production of ethanol by a Z. mobilis strain of the invention is described.

For production of ethanol, recombinant xylose-utilizing Z. mobilis having a Group I xylose isomerase chimeric gene is brought in contact with medium that contains mixed sugars including xylose. Typically the medium contains mixed sugars including arabinose, xylose, and glucose. The medium may contain biomass hydrolysate that includes these sugars that are derived from treated cellulosic or lignocellulosic biomass.

When the mixed sugars concentration is high such that growth is inhibited, the medium includes sorbitol, mannitol, or a mixture thereof as disclosed in U.S. Pat. No. 7,629,156. Galactitol or ribitol may replace or be combined with sorbitol or mannitol. The Z. mobilis grows in the medium where fermentation occurs and ethanol is produced. The fermentation is run without supplemented air, oxygen, or other gases (which may include conditions such as anaerobic, microaerobic, or microaerophilic fermentation), for at least about 24 hours, and may be run for 30 or more hours. The timing to reach maximal ethanol production is variable, depending on the fermentation conditions. Typically, if inhibitors are present in the medium, a longer fermentation period is required. The fermentations may be run at temperatures that are between about 30° C. and about 37° C., at a pH of about 4.5 to about 7.5.

The present Z. mobilis may be grown in medium containing mixed sugars including xylose in laboratory scale fermenters, and in scaled up fermentation where commercial quantities of ethanol are produced. Where commercial production of ethanol is desired, a variety of culture methodologies may be applied. For example, large-scale production from the present Z. mobilis strains may be produced by both batch and continuous culture methodologies. A classical batch culturing method is a closed system where the composition of the medium is set at the beginning of the culture and not subjected to artificial alterations during the culturing process. Thus, at the beginning of the culturing process the medium is inoculated with the desired organism and growth or metabolic activity is permitted to occur adding nothing to the system. Typically, however, a “batch” culture is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase are often responsible for the bulk of production of end product or intermediate in some systems. Stationary or post-exponential phase production can be obtained in other systems.

A variation on the standard batch system is the Fed-Batch system. Fed-Batch culture processes are also suitable for growth of the present Z. mobilis strains and comprise a typical batch system with the exception that the substrate is added in increments as the culture progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH and the partial pressure of waste gases such as CO₂. Batch and Fed-Batch culturing methods are common and well known in the art and examples may be found in Biotechnology: A Textbook of Industrial Microbiology, Crueger, Crueger, and Brock, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36, 227, (1992), herein incorporated by reference.

Commercial production of ethanol may also be accomplished with a continuous culture. Continuous cultures are open systems where a defined culture medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in log phase growth. Alternatively, continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added, and valuable products, by-products or waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural and/or synthetic materials as is known to one skilled in the art.

Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by medium turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to medium being drawn off must be balanced against the cell growth rate in the culture. Methods of modulating nutrients and growth factors for continuous culture processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

Particularly suitable for ethanol production is a fermentation regime as follows. The desired Z. mobilis strain of the present invention is grown in shake flasks in semi-complex medium at about 30° C. to about 37° C. with shaking at about 150 rpm in orbital shakers and then transferred to a 10 L seed fermentor containing similar medium. The seed culture is grown in the seed fermentor anaerobically until OD₆₀₀ is between 3 and 6, when it is transferred to the production fermentor where the fermentation parameters are optimized for ethanol production. Typical inoculum volumes transferred from the seed tank to the production tank range from about 2% to about 20% v/v. Typical fermentation medium contains minimal medium components such as potassium phosphate (1.0-10.0 g/l), ammonium sulfate (0-2.0 g/l), magnesium sulfate (0-5.0 g/l), a complex nitrogen source such as yeast extract or soy based products (0-10 g/l). A final concentration of about 5 mM sorbitol or mannitol is present in the medium. Mixed sugars including xylose and at least one additional sugar such as glucose (or sucrose), providing a carbon source, are continually added to the fermentation vessel on depletion of the initial batched carbon source (50-200 g/l) to maximize ethanol rate and titer. Carbon source feed rates are adjusted dynamically to ensure that the culture is not accumulating glucose in excess, which could lead to build up of toxic byproducts such as acetic acid. In order to maximize yield of ethanol produced from substrate utilized, biomass growth is restricted by the amount of phosphate that is either batched initially or that is fed during the course of the fermentation. The fermentation is controlled at pH 5.0-6.0 using caustic solution (such as ammonium hydroxide, potassium hydroxide, or sodium hydroxide) and either sulfuric or phosphoric acid. The temperature of the fermentor is controlled at 30° C.-35° C. In order to minimize foaming, antifoam agents (any class-silicone based, organic based etc) are added to the vessel as needed. An antibiotic, for which there is an antibiotic resistant marker in the strain, such as kanamycin, may be used optionally to minimize contamination.

Any set of conditions described above, and additionally variations in these conditions that are well known in the art, are suitable conditions for production of ethanol by a xylose-utilizing recombinant Zymomonas strain.

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

General Methods

The meaning of abbreviations is as follows: “kb” means kilobase(s), “bp” means base pairs, “nt” means nucleotide(s), “hr” means hour(s), “min” means minute(s), “sec” means second(s), “d” means day(s), “L” means liter(s), “ml” or “mL” means milliliter(s), “4” means microliter(s), “μg” means microgram(s), “ng” means nanogram(s), “mg” means milligram(s), “mM” means millimolar, “μM” means micromolar, “nm” means nanometer(s), “μmol” means micromole(s), “pmol” means picomole(s), “Cm” means chloramphenicol, “Cm^(r)” means chloramphenicol resistant, “Cm^(s)” means chloramphenicol sensitive, “Sp^(r)” means spectinomycin resistance, “Sp^(s)” means spectinomycin sensitive, “XI” is xylose isomerase, “XK” is xylulokinase, “TAL” is transaldolase, “TKT” is transketolase, “RM” means rich medium containing 10 g/L yeast extract plus 2 g/L KH₂PO₄, “MM” means mating medium containing 10 g/L yeast extract, 5 g/L tryptone, 2.5 g/L (NH₄)₂SO₄ and 0.2 g/L KH₂PO₄.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience, Hoboken, N.J. (1987).

Example 1 Construction of Chimeric Xylose Isomerase Genes and Assembly of Double Crossover Suicide Vectors

Constructs were made for integration and expression in Zymomonas mobilis of the coding regions for xylose isomerases from Actinoplanes missouriensis (AMxylA), Lactobacillus brevis (LBxylA), and Escherichia coli (ECxylA). The coding sequences were optimized for expression in Z. mobilis according to the codon bias of Z. mobilis ZM4 and synthesized by GenScript Corporation (Piscataway, N.J.). Each was cloned into pUC57 at the EcoRV site and provided as the plasmids pUC57-AMxylA (with codon optimized AMxylA coding region SEQ ID NO:308), pUC57-LBxylA (with codon optimized LBxylA coding region SEQ ID NO:309), and pUC57-ECxylA (with codon optimized ECxylA coding region SEQ ID NO:310). The optimized xylA coding sequences encode the native xylose isomerases (XIs).

The xylA coding sequences were constructed into chimeric genes with the structure of P_(gap)-xylA-araD3′UTR by linking with a 305 bp promoter from the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene (P_(gap); SEQ ID NO:311) and a 166 bp terminator from the E. coli L-ribulose 5 phosphate 4-epimerase gene (araD3′UTR; SEQ ID NO:312).

For this purpose, P_(gap) and araD3′UTR overlapping fragments were synthesized by PCR. One PCR reaction consisted of 50 μL AccuPrime Pfx SuperMix (Invitrogen, Carlsbad, Calif.), 1 μL of 40 ng/μL pARA354 as template (SEQ ID NO:313), and 1 μL of 10 μM forward and reverse primers. Plasmid pARA354 (SEQ ID NO:313) is described in commonly owned and co-pending U.S. patent application Ser. No. 12/796,025, which is herein incorporated by reference, and is a pBS SK(+) vector that includes a P_(gap)-ara BAD operon, which is the P_(gap) promoter adjacent to coding regions for araB, araA, and araD (encoding the proteins L-ribulose kinase, L-arabinose isomerase, and L-ribulose-5-phosphate-4-epimerase, respectively) from E. coli. The operon includes the 3′ untranslated region (UTR) that is 3′ to the araD coding region. pARA354 is described further below. Reactions were carried out on an Eppendorf Mastercycler (Hemburg, Germany), following a hot start PCR program with 30 cycles of denaturing at 95° C. for 30 sec/annealing at 58° C. (56° C. for araD3′UTR) for 30 sec/extension at 68° C. for 2 min.

Primers ara98 and ara120 (SEQ ID NOS:314 and 315) produced a P_(gap)-AM overlapping fragment. Primers ara98 and ara121 (SEQ ID NOS: 314 and 316) produced a P_(gap)-EC overlapping fragment. Primers ara98 and ara122 (SEQ ID NOS: 314 and 317) produced a P_(gap)-LB overlapping fragment. In addition to the 305 bp P_(gap) sequence, all three P_(gap) overlapping PCR fragments included a 17 bp 5′ sequence to add StuI and SpeI sites and a 22 bp 3′ sequence to match the first 22 nucleotides of their counterpart xylA coding sequence, that were provided in the primers.

Primer ara96 and ara97 (SEQ ID NOS: 318 and 319) produced a 210-bp araD3′UTR overlapping fragment. It included a 24 bp 5′ sequence with an XbaI site at the end, and a 20 bp 3′ sequence providing EcoRI, HindII, and FseI sites, in addition to the 166-bp araD3′UTR. Similar PCR was also conducted to synthesize xylA overlapping fragments, but annealing temperature was lowered to 55° C. In these reactions, a 1,229 bp AMxylA overlapping fragment was amplified from pUC57-AMxylA using primers ara114 and ara115 (SEQ ID NOs:320 and 321). A 1,367-bp ECxylA overlapping fragment was amplified from pUC57-ECxylA using primers ara116 and ara117 (SEQ ID NOs:322 and 323). A 1,394-bp LBxylA overlapping fragment was amplified from pUC57-LBxylA using primers ara118 and ara119 (SEQ ID NOs:324 and 325). All xylA overlapping fragments have an 18-bp 5′ sequence matching the last 18 nucleotides of P_(gap), a 24-bp 3′ sequence providing an XbaI site and matching the first 18 nucleotides of araD3′UTR, as well as a xylA coding sequence between them. The P_(gap), araD3′UTR, and xylA overlapping fragments were confirmed by running 5 μL of each PCR sample on an agarose gel, and then purified by using QIAquick PCR Purification Kit (Qiagen, Valencia, Calif.).

Overlapping fragments were linked together by overlapping PCR. The first overlapping PCR was assembled to include 50 μL AccuPrime Pfx SuperMix, 1 μL of 20 ng/μL P_(gap) overlapping fragment, 2 μL of 10 ng/μL counterpart xylA overlapping fragment, and 1 μL of 10 μM forward and reverse primers as follows. Reaction was conducted by following a hot start PCR program with 30 cycles of denaturing at 95° C. for 30 sec/annealing at 55° C. for 30 sec/extension at 68° C. for 2 min. As a result, a P_(gap)-AMxylA fragment was synthesized from P_(gap)-AM and AMxylA fragments using primers ara98 and ara115 (SEQ ID NOs:314 and 321); a P_(gap)-ECxylA fragment was synthesized from P_(gap)-EC and ECxylA fragments using primers ara98 and ara117 (SEQ ID NOs:314 and 323); and a P_(gap)-LBxylA fragment was synthesized from P_(gap)-LB and LBxylA fragments using primers ara98 and ara119 (SEQ ID NOs:314 and 325).

These P_(gap)-xylA fragments were confirmed by running 5 μL of each PCR sample on an agarose gel, and then purified by using QIAquick PCR Purification Kit.

A second overlapping PCR was assembled similarly to above. It included 50 μL AccuPrime Pfx SuperMix, 1 μL of 20 ng/μL araD3′UTR overlapping fragment, 2 μL of 10 ng/μL P_(gap)-xylA fragment, 1 μL of 10 μM primer ara97 (SEQ ID NO:319), and 1 μL of 10 μM primer ara98 (SEQ ID NO:318). Reaction was carried out for 30 cycles of denaturing at 95° C. for 30 sec/annealing at 56° C. for 30 sec/extension at 68° C. for 2.5 min. Five microliters of the resulting PCR product were inspected on an agarose gel. The reactions containing P_(gap)-AMxylA and P_(gap)-LBxylA produced a 1,714-bp chimeric AMxylA operon fragment (P_(gap)-AMxylA-araD3′UTR) and a 1,879-bp chimeric LBxylA operon fragment (P_(gap)-LBxylA-araD3′UTR), respectively. In both chimeric gene fragments, the first 17 nucleotides provide StuI and SpeI sites while the last 35 nucleotides contain FesI, HindIII, and EcoI sites. The PCR reaction containing P_(gap)-ECxylA failed to generate the chimeric ECxylA fragment (P_(gap)-ECxylA-araD3′UTR).

The chimeric genes containing AMxylA and LBxylA were each ligated into a double crossover (DCO) vector named pARA354 (SEQ ID NO:313) that was described in commonly owned and co-pending U.S. patent application Ser. No. 12/796,025. pARA354 is pBS SK(+) derived plasmid (a Bluescript plasmid; Stratagene), used as a suicide vector since pBS vectors cannot replicate in Zymomonas, containing a P_(gap)-araBAD operon as described above and DCO homologous recombination fragments to direct integration of a bounded fragment into the IdhA locus of the Zymomonas genome. The two IdhA DNA fragments of pARA354 for DCO, LDH-L and LDH-R, were synthesized by PCR using Z. mobilis DNA as template. The reaction used AccuPrime Mix and followed a standard PCR procedure. The LDH-L DNA fragment was synthesized using forward primer ara20 (SEQ ID NO:326) and reverse primer ara21 (SEQ ID NO:327). The resulting product was an 895-bp DNA fragment including sequence 5′ to the IdhA coding region and nucleotides 1-493 of the IdhA coding region, with a 5′ SacI site and a 3′ SpeI site (SEQ ID NO:328). The LDH-R DNA fragment was synthesized using forward primer ara22 (SEQ ID NO:329) and reverse primer ara23 (SEQ ID NO:330). The resulting product was a 1169 bp fragment including nucleotides 494-996 of the IdhA coding region and sequence 3′ to the IdhA coding region, with a 5′ EcoRI site and a 3′ NotI site (SEQ ID NO:331). Since LDH-L and LDH-R contained the first 493 base pairs and the remaining 503 base pairs of the IdhA coding sequence, respectively, pARA354 was designed to direct insertion of a DNA fragment into the IdhA coding sequence of Z. mobilis between nucleotides #493 and #494 by crossover recombination

pARA354 contains an f1(+) origin and an ampicillin resistance gene for plasmid propagation in E. coli. In addition, between the LDH-L and LDH-R homologous recombination fragments in pARA354 is the aadA marker (for spectinomycin resistance) bounded by wild type LoxP sites (LoxPw-aadA-LoxPw fragment; SEQ ID NO:307) and a P_(gap)-araBAD operon.

The PCR fragments containing AMxylA and LBxylA chimeric genes were each digested with SpeI and EcoRI, subjected to agarose gel electrophoresis, and purified by using QIAquick Gel Purification Kit (Qiagen). At the same time, pARA354 was also digested with SpeI and EcoRI to drop out the P_(gap)araBAD operon. The EcoRI-pARA354-SpeI plasmid backbone (6,023 bp) was isolated by agarose gel electrophoresis, and purified by using a QIAquick Gel Purification Kit. The chimeric AMxylA and LBxylA genes were each constructed into the pARA354 backbone in 15 μL standard ligation reactions that included 5 μL of the digested AMxylA or LBxylA chimeric gene fragment, 2 μL of the digested pARA354 backbone, 3 μL 5× ligase buffer, and 1 μL T4 DNA ligase (Invitrogen), resulting in a 7,714-bp DCO plasmid pARA355 and a 7,879-bp DCO plasmid pARA356, respectively. Both plasmids were propagated in DH5α E. coli cells and prepared by using QIAprep Spin Miniprep Kit (Qiagen).

To construct a chimeric ECxylA gene in the DCO vector, the P_(gap)-ECxylA overlapping fragment produced in the first overlapping PCR was digested with SpeI and XbaI, subjected to agarose gel electrophoresis, and purified by using QIAquick Gel Purification Kit. At the same time, pARA355 was digested with SpecI and XbaI. The XbaI-pARA355-SpeI plasmid backbone (6,220 bp) was isolated by agarose gel electrophoresis, and purified by using QIAquick Gel Purification Kit. The P_(gap)-ECxylA fragment was assembled into the pARA355 backbone in a 15 μL standard ligation reaction as described above, including 5 μL of the digested P_(gap)-ECxylA fragment and 2 μL of the digested pARA355 backbone fragment. The resultant 7,852 bp DCO plasmid pARA357 was propagated in DH5α E. coli cells and prepared by using QIAprep Spin Miniprep Kit.

Example 2 Integration of Chimeric AMxylA, LBxylA, and ECxylA Genes into Zymomonas mobilis Strain ZW641

Effects of expressing the A. missourinesis, L. brevis or E. coli XI in a xylose-utilizing Zymomonas mobilis were assayed using strain ZW641. Preparation of the ZW641 strain is described in Example 1 of U.S. Pat. No. 7,741,119, which is incorporated herein by reference. Strain X13L3 described therein was later renamed ZW641. ZW641 was prepared by sequentially integrating the two operons P_(gap)xylAB and P_(gap)taltkt, along with the chloramphenicol resistance selectable marker, into the genome of Zymomonas mobilis ZW1 (ATCC #31821). Transformants were further adapted for xylose utilization by growth in xylose-containing medium.

In the ZW641 integrated P_(gap)xylAB and P_(gap)taltkt operons the xylA, xylB, tal, and tkt coding regions are from E. coli genes. Though ZW641 has all four genes necessary for xylose metabolism, xylose utilization is not optimal. Thus a background level of xylose utilization in ZW641 could potentially be improved by expressing an additional XI gene.

Competent cells of the ZW641-1A strain (a ZW641 isolate) were prepared by growing seed cells overnight in MRM3G5 (1% yeast extract, 15 mM KH₂PO₄, 4 mM MgSO₄, and 50 g/L glucose) at 30° C. with 150 rpm shaking, to an OD₆₀₀ value near 5. The OD₆₀₀ value was measured using a Shimadzu UV-1200 Spectrophotometer (Kyoto, Japan). Cells were harvested and resuspended in fresh medium to an OD₆₀₀ value of 0.05. The cells were grown under the same conditions to early to middle log phase (OD₆₀₀ near 0.5). Cells were harvested and washed twice with ice-cold water and then once with ice-cold 10% glycerol. The resulting competent cells were collected and resuspended in ice-cold 10% glycerol to an OD₆₀₀ value near 100. Since transformation of Z. mobilis requires non-methylated DNA, DCO plasmids pARA355, pARA356, and pARA357 were each transformed into E. coli SCS110 competent cells (Stratagene, La Jolla, Calif.). For each transformation, one colony of transformed cells was grown in 10 mL LB-Amp 100 (LB broth containing 100 mg/L ampicillin) overnight at 37° C. DNA was prepared from the 10 mL culture, using QIAprep Spin DNA Miniprep Kit (Qiagen).

Approximately 1 μg non-methylated plasmid DNA was mixed with 50 μL ZW641-1A competent cells in a 1 mM Electroporation Cuvette (VWR, West Chester, Pa.). The plasmid DNA was electroporated into the cells at 2.0 KV using a BT720 Transporater Plus (BTX-Genetronics, San Diego, Calif.). Transformed cells were recovered in 1 mL MMG5 medium (10 g/L glucose, 10 g/L yeast extract, 5 g/L tryptone, 2.5 g/L (NH₄)₂SO₄, 2 g/L K₂HPO₄, and 1 mM MgSO₄) for 4 hours at 30° C. and grown on MMG5-Spec250 plates (MMG5 with 250 mg/L spectinomycin and 15 g/L agar) for 3 days at 30° C., inside an anaerobic jar with an AnaeroPack (Mitsubishi Gas Chemical, New York, N.Y.). About 20 spectinomycin-resistant colonies were obtained for each transformation. These colonies were streaked onto a fresh MMG5-Spec250 plate and their growth under the same conditions as described above indicated that the chimeric xylA gene/Spec-R construct had been integrated into the genome of ZW641. Integration was analyzed by PCR. One reaction included 25 μL PCR SuperMix (Invitrogen), 0.5 μL 10 μM forward primer and reverse primer (as specified below), and a small amount of Z. moblis cells from the colonies. Reaction was carried out on an Eppendorf Mastercycler, following a hard start PCR program with 35 cycles of denaturing at 94° C. for 45 sec/annealing at 55° C. for 45 sec/extension at 72° C. for 1.5 min. Reactions were examined by running 5 μL on an agarose gel. When ara46 and ara43 primers (SEQ ID NOs:332 and 333) were used in the first inspection, a 1,521-bp PCR product was amplified from most colonies. This product spans from the aadA coding region of the Spec-R marker in the plasmids to a Z. mobilis genomic sequence downstream of the LDH-R fragment, demonstrating the integration events mediated by the LDH-R fragment. When forward-reverse primer pairs were ara45-ara120 (SEQ ID NOs:334 and 315), ara45-ara122 (SEQ ID NOs:334 and 317), and ara45-ara121 (SEQ ID NOs:334 and 316) in the second inspection, a 1,289-bp PCR product was amplified from the colonies of ZW641-ara355, ZW641-ara356, and ZW641-ara357, respectively. These products span from a Z. mobilis genomic sequence upstream of the LDH-L fragment to AMxylA in pARA355, LBxylA in pARA356, or ECxylA in pARA357. These demonstrated the integration events mediated by the LDH-L fragment. Therefore, PCR evidence confirmed that the chimeric xylA operon/Spec-R construct had been integrated into the genome of ZW641-1A. The ZW641-1A cells transformed with pARA355, pARA356, and pARA357 were named as ZW641-ara355, ZW641-ara356, and ZW641-ara357, respectively.

Example 3 Characterization of AMxylA, LBxylA, and ECxylA Expression in Zymomonas mobilis Strain ZW641

ZW641 has a copy of the native E. coli xylA coding region, which is expressed at a low level. Strains ZW641-ara355, ZW641-ara356, and ZW641-ara357 each contain an additional copy of a codon-optimized xylA coding region: AMxylA, LBxylA, and ECxylA, respectively. Enhanced xylose utilization, ethanol production, and growth in xylose were assayed for the strains.

To examine the growth of these new strains in media containing xylose and compare them with the parent strain ZW641-1A, two strains (#1 and #2) of each of ZW641-ara355, ZW641-ara356, and ZW641-ara357 from the MMG5-Spec250 plates described in the previous example were re-streaked onto a MM×5 plate (the same medium except glucose is replaced by xylose). ZW641 was also streaked onto the plate as a control. Cells on the plate were grown for 6 days at 30° C. inside an anaerobic jar with an AnaeroPack. Growth was observed for all three sets of the new strains, but not for the ZW641 control. The #1 and #2 strains of ZW641-ara355, which contain an additional copy of AMxylA, showed significantly more growth on the xylose medium than ZW641-ara356 and ZW641-ara357 strains.

To quantitatively measure the growth in xylose, these 7 strains were subjected to a 96-hour growth assay. In the assay, cells from each strain were grown overnight in 3 mL MRM3G5 in a 30° C. 150 rpm shaker. Cells were harvested, washed with MRM3×10 (same as MRM3G5 but 50 g/L glucose was replaced by 100 g/L xylose), and resuspended in MRM3×10 to have a starting OD₆₀₀ value near 0.1. Twenty-five milliliters of the suspension was placed in a 50 mL screw capped VWR centrifuge tube and grown at 30° C. with 150 rpm shaking for a 96-hour time course. During the time course, OD₆₀₀ value was measured at 0-, 4-, 24-, 48-, 72-, and 96-hours. The results plotted as growth curves in FIG. 4 show that the second copy of xylA indeed enhanced the growth of the engineered strains in xylose containing medium. When comparing between those strains containing the second copy of xylA, ZW641-ara355 grew significantly faster than ZW641-ara357. It had a cell density almost twice as high as ZW641-ara357 after 96 hours of growth. This result indicates that the xylose isomerase encoded by AMxylA may function much better than the xylose isomerase encoded by ECxylA. ZW641-ara356 grew similarly to or slightly slower than ZW641-ara357, indicating that the xylose isomerase encoded by LBxylA may not function better than the xylose isomerase encoded by ECxylA.

During the time course, 1 mL samples of the ZW641, ZW641-ara355-1, ZW641-ara356-2, and ZW641-ara357-2 cultures were collected at the 72-hour point. They were centrifuged at 10,000×g to remove cells. The supernatant was filtered through a 0.22 μm Costar Spin-X Centrifuge Tube Filter and analyzed by running through a BioRad Aminex HPX-A7H ion exclusion column with 0.01 N H₂SO₄ in a speed of 0.6 mL/min at 55° C. on an Agilent 1100 HPLC system to determine ethanol and xylose concentrations. The results given in Table 5 show that, comparing to the basal level xylose utilization and ethanol production in ZW641, AMxylA in ZW641-ara355 significantly promoted xylose consumption and had increased ethanol production by more than 3.5 fold. ECxylA in ZW641-ara357 slightly increased xylose metabolism and ethanol production, while LBxylA in ZW641-ara356 offered the smallest increase in xylose utilization and did not cause a detectable change in ethanol production. These results agree with previous observations on the growth and suggest that the difference in growth between the strains was caused by the difference in xylose metabolism, which may result from a difference in xylose isomerase activity.

TABLE 5 Cell growth, xylose consumption, and ethanol production after 72 hours culturing at 30° C. in MRM3X10. Additional XI Growth Ethanol Xylose* Strain source (OD₆₀₀) (g/L) (g/L) ZW641 none 0.46 1.9 93.1 ZW641-ara355-1 A. missourinesis 1.14 6.8 81.7 ZW641-ara356-2 L. brevis 0.53 1.9 92.9 ZW641-ara357-2 E. coli 0.61 2.2 91.7 MRM3X10⁺ na^(#) na 0.0 95.8 *xylose remaining in the medium ⁺starting media ^(#)na: not applicable

To determine whether xylose isomerase enzymes encoded by AMxylA, LBxylA, and ECxylA have different activities, ZW641, ZW641-ara355-1, ZW641-ara356-2, and ZW641-ara357-2 were grown overnight in MRM3G5 on a 30° C. 150 rpm shaker. Cells were collected from 2 mL cultures by 10,000×g centrifugation, washed with ice-cold Protein Extraction Buffer (10 mM triethanolamine hydrochloride, pH8.0, 10 mM MgSO₄, 1 mM DTT, and 5% glycerol), resuspended in 500 μL ice-cold Protein Extraction Buffer, and subjected to 3 minutes sonication for 3 times at setting 7 by using a Misonix Sonicator 4000 with microplate horn (Qsonica, Newtown, Conn.). Cell debris was removed by 10,000× g centrifugation. Supernatants were kept as protein extracts. Their protein concentrations were measured by using Coomassie Plus Protein Assay Reagent (Pierce, Rockford, Ill.), and xylose isomerase activities were measured by a modified Cysteine-Carboazole method. A 100 μL Cysteine-Carboazole assay reaction contained 10 mM triethanolamine hydrochloride buffer, pH7.0, 10 mM MgSO₄, 25 mM D-xylose, and 30 μg extracted protein. After incubation at 32° C. for 15 minutes, the reaction was stopped by adding 25 μl 50% trichloroacetic acid. Then, 3 ml ice-cold 75% sulphuric acid, 100 μl 2.4% cysteine hydrochloride solution, and 100 μl 0.12% carbazole ethanolic solution were sequentially added into the reaction. The mixture was kept at room temperature for 10 minutes. OD₅₄₀ value was measured on a Shimadzu UV-1200 Spectrophotometer. The corresponding D-xylulose concentration was determined based on a standard curve of D-xylulose concentration vs. OD₅₄₀ value. The standard curve was developed by carrying out Cysteine-Carboazole assays containing various amounts of D-xylulose but no D-xylose and protein. Finally, one unit of xylose isomerase enzyme was defined as the activity required to produce one micromole of D-xylulose in the reaction. Specific activity was calculated as unit per milligram of protein. In the assay, background OD₅₄₀ of D-xylose was measured in a blank reaction without protein. This background was subtracted from the original OD₅₄₀ reading prior to calculation of activity. Each assay was repeated 3 times. The results of the Cysteine-Carboazole assay are graphed in FIG. 5 showing specific activities of xylose isomerase in the protein extracts of ZW641, ZW641-ara355-1, ZW641-ara356-2, and ZW641-ara357-2. Each activity bar represents an average of three parallel reactions with a standard deviation calculated based on them. This result demonstrates that expression of the second copy of xylA in ZW641 introduced additional xylose isomerase activity into cells. AMxylA in ZW641-ara355, LBxylA in ZW641-ara356, and ECxylA in ZW641-ara357 increased XI activity by approximately 20-fold, 3 fold, and 4 fold, respectively. Since these three xylA genes are constructed in ZW641 by the same approach, the difference in the xylose isomerase specific activities in the cell extracts suggests that the xylose isomerase from A. missouriensis functions much better than xylose isomerases from L. brevis and E. coli. In fact, the A. missouriensis XI presented a specific activity of about 2.3, which was 6 times higher than L. brevis XI and 5 times higher than E. coli XI. Therefore, by examining cell growth, xylose metabolism, and XI activity, this example has identified A. missouriensis XI as an excellent xylose isomerase for improving xylose utilization in xylose-utilizing Z. mobilis strains.

Example 4 Structural Analysis of Xylose Isomerase Enzymes

A collection of available protein sequences that are potentially xylose isomerases was prepared by first identifying a set of seed sequences that are known to have xylose isomerase (XI) activity. The seed sequences were retrieved as xylose isomerases from the SWISSPROT database, which contains protein sequences that have high confidence functional annotations. There were 180 XI seed sequences retrieved from SWISSPROT (Swiss Institute of Bioinformatics): SEQ ID NOs:2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, 142 and 148-306. These seed sequences were then used to search the NCBI (National Center for Biotechnology Information, Bethesda, Md.) non-redundant (nr) comprehensive protein database as a group of multiple query sequences in the blastall wrapper of BLAST. A total of 444 sequences were identified to form a set that can be described as the sequence space of XI activity proteins.

Clustering based on sequence identity and molecular phylogenetic analysis using the PHYLIP neighbor joining algorithm (as implemented in PHYLIP (Phylogeny Inference Package) version 3.5c (Felsenstein (1989) Cladistics 5:164-166) showed that the above generated sequence space for XI activity separated into two groups referred to as Group I and Group II as shown in FIG. 2. The Group I set consisted of 82 sequences (SEQ ID NOs that are even numbers between 2 and 130 and 131-147), 21 of which were seeds (SEQ ID NOs:2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, and 142). Similarly, the Group II set consisted of 351 members, 159 of which were seeds (SEQ ID NOs:148-306). As shown in FIG. 2, the L. brevis and E. coli XI proteins belong to Group II while the A. missouriensis XI protein belongs to Group I.

The following process was followed in forming the phylogenetic groups:

Starting with the 444 XI sequences:

Step 1 establishing 70% identity groups:

The longest sequence out of the 444 sequences was designated as the first master. Other sequences of the 444 sequences that have 70% or more sequence identity to the first master were grouped to form the first ref70 cluster (A). Out of the remaining sequences, the longest sequence was designated the second master and used create the second ref70 cluster similarly (B). The grouping process was continued until every sequence was in a cluster. Some of the clusters were singletons.

Step 2 merging at 70% threshold:

For every pair of clusters A and B, if a third of the sequences in A are related to sequences in B by 70% sequence identity or more and vice-versa, clusters A and B were merged. This process was continued until there were no pairs that could be merged using the 70% identity threshold.

Step 3 merging at 50% threshold:

The same process as in step 2 was followed but using a sequence identity threshold of 50%.

Step 4 merging at 30% threshold:

The same process as in step 2 was followed but using a sequence identity threshold of 30%.

Group I represents a 50% threshold identity cluster. Group II represents a separate 50% threshold identity cluster.

There were 11 sequences that were not unambiguously assigned to Group I or Group II since they did not cluster with either group at the 50% threshold of identity.

FIG. 3 shows a phylogenetic tree for the Group I XIs with specific genera labeled. Group I includes XI proteins from Arthrobacter, Streptomyces, Thermus, Thermobaculum, Herpetosiphon, Acidobacteria, Roseiflexus, Meiothermus, Deinococcus, Meiothermus, Stackebrandtia, Kribbella, Xylanimonas, Nocardiopsis, Catenulispora, Streptosporangium, Geodermatopyilus, Actinosynnema, Saccharomonospora, Acicothermus, Tthermobifida, Nocardiodes, janibacter, Mycobacterium, Leifsonia, Clavibacter, Micromonospora, Salinispora, Cellulomonas, jonesia, Nakamurella, Actinomyces, Mobiluncus, Brachybacterium, Beutengergai, Frankia, and Actinobacterium.

Discriminating Between Group I and Group II: Method 1

Discrimination between Group I members and Group II members was performed by GroupSim analysis (Capra and Singh (2008) Bioinformatics 24: 1473-1480). The GroupSim method identifies amino acid residues that determine a protein's functional specificity. In a multiple sequence alignment (MSA) of a protein family whose sequences are divided into multiple groups, amino acid residues that distinguish between the functional groups of sequences can be identified. The method takes a multiple sequence alignment (MSA) and known specificity groupings as input, and assigns a score to each amino acid position in the MSA. Higher scores indicate a greater likelihood that an amino acid position is a specificity determining position (SDP).

GroupSim analysis performed on the MSA of XI sequences that were divided into Group I and Group II by the phylogenetic analysis above identified highly discriminating positions. Listed in Table 6 are positions (Pos) having scores greater than or equal to 0.9, where a perfect score of 1.0 would indicate that all proteins within the group have the listed amino acid in the specified position and between groups the amino acid would always be different. The “residue” column gives the amino acid(s) in single letter code for the position in Group I proteins vs in Group II proteins (separated by a bar: I). The amino acid position number in column 2 is for the representative Group I protein P12581 which is the XI from Actinoplanes missourinesis. The amino acid position number in column 3 is for the representative Group II protein P19148 which is the XI Thermoanaerobacterium thermosulfurigenes (SEQ ID NO:267)

TABLE 6 Highly discriminating amino acid positions for Group I and Group II XIs from GroupSim analysis. Pos in P12851 Pos in (member Pos in P19148 Residue alignment of Group I) (member of Group II) (Group I|Group II) 391 226 277 L|H 388 223 274 M|L 341 191 242 I|Q 345 195 246 TSV|D 228 88 139 MTG|RW 462 290 337 H|NM 386 221 272 ED|ATG 407 242 293 FVL|GCW 408 243 294 H|SNGL 343 193 244 LFM|D 422 256 308 Q|TIVLMYH 377 213 264 G|KNSEALRQ 460 288 335 PYAS|VG 415 249 301 Q|DHNRSA Discriminating Between Group I and Group II: Method 2

An alternative structure/function characterization of the groups of the xylose isomerase family of enzymes was performed using the HMMER software package (the theory behind profile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235:1501-1531), following the user guide which is available from HMMER (Janelia Farm Research Campus, Ashburn, Va.).

Using a multiple sequence alignment of the 21 seed sequences in Group I (SEQ ID NOs:2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, and 142), a profile Hidden Markov Model (HMM) was created for representing Group I members. As stated in the user guide, Profile HMMs are statistical models of multiple sequence alignments. They capture position-specific information about how conserved each column of the alignment is, and which amino acid residues are most likely to occur at each position. Thus HMMs have a formal probabilistic basis. Profile HMMs for a large number of protein families are publicly available in the PFAM database (Janelia Farm Research Campus, Ashburn, Va.).

The Profile HMM was built as follows:

Step 1. Build a Sequence Alignment

The 21 seed sequences (sequences with high confidence annotation) that are in Group I were aligned using Clustal W with default parameters.

Step 2. Build a Profile HMM

The hmmbuild program was run on the set of aligned sequences using default parameters. hmmbuild reads the multiple sequence alignment file, builds a new Profile HMM, and saves the Profile HMM to file. Using this program an un-calibrated profile was generated from the multiple alignment for the set of seed sequences described above.

The following information based on the HMMER software user guide gives some description of the way that the hmmbuild program prepares a Profile HMM. A Profile HMM is capable of modeling gapped alignments, e.g. including insertions and deletions, which lets the software describe a complete conserved domain (rather than just a small ungapped motif). Insertions and deletions are modeled using insertion (I) states and deletion (D) states. All columns that contain more than a certain fraction x of gap characters will be assigned as an insert column. By default, x is set to 0.5. Each match state has an I and a D state associated with it. HMMER calls a group of three states (M/D/I) at the same consensus position in the alignment a “node”. These states are interconnected with arrows called state transition probabilities. M and I states are emitters, while D states are silent. The transitions are arranged so that at each node, either the M state is used (and a residue is aligned and scored) or the D state is used (and no residue is aligned, resulting in a deletion-gap character, ‘-’). Insertions occur between nodes, and I states have a self-transition, allowing one or more inserted residues to occur between consensus columns.

The scores of residues in a match state (i.e. match state emission scores), or in an insert state (i.e. insert state emission scores) are proportional to Log_(—)2 (p_x)/(null_x). Where p_x is the probability of an amino acid residue, at a particular position in the alignment, according to the Profile HMM and null_x is the probability according to the Null model. The Null model is a simple one state probabilistic model with pre-calculated set of emission probabilities for each of the 20 amino acids derived from the distribution of amino acids in the SWISSPROT release 24.

State transition scores are also calculated as log odds parameters and are propotional to Log_(—)2 (t_x). Where t_x is the probability of transiting to an emitter or non-emitter state.

Step 3. Calibrate the Profile HMM

The Profile HMM was read using hmmcalibrate which scores a large number of synthesized random sequences with the Profile (the default number of synthetic sequences used is 5,000), fits an extreme value distribution (EVD) to the histogram of those scores, and re-saves the HMM file now including the EVD parameters. These EVD parameters (μ and λ) are used to calculate the E-values of bit scores when the profile is searched against a protein sequence database. hmmcalibrate writes two parameters into the HMM file on a line labeled “EVD”: these parameters are the μ (location) and λ (scale) parameters of an extreme value distribution (EVD) that best fits a histogram of scores calculated on randomly generated sequences of about the same length and residue composition as SWISS-PROT. This calibration was done once for the Profile HMM.

The calibrated profile HMM for the Group I set is provided as Table 1 in the appendix as a Group I profile HMM Excel chart. The Profile HMM is provided in a chart that gives the probability of each amino acid occurring at each position in the amino acid sequence. The highest probability is highlighted for each position. Table 7 shows a few lines of the Group I Profile HMM.

TABLE 7 A portion of the Group I Profile HMM. HMM A C D E F G H I K L M m−>m m−>i m−>d i−>m i−>i d−>m d−>d b−>m m−>e −462 * −1868 1 −1476 −1441 −2702 −2503 −679 −2424 −1861 −88 −2024 240 4620 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 — −24 −6482 −7525 −894 −1115 −701 −1378 −462 * 2 421 −976 −556 −40 −1303 −1252 −182 −920 65 −1119 1573 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 3 −926 −719 −2839 −2316 1366 −2394 −664 122 −1975 1177 430 — −149 −500 −233 43 −381 399 106 −626 210 −466 −720 — −21 −6672 −7714 −894 −1115 −701 −1378 * * HMM N P Q R S T V W Y 1 −2293 −2670 −1975 −1997 −1977 −1568 −381 −1705 −1336 — 275 394 45 96 359 117 −369 −294 −249 — 2 1057 −1439 138 −354 1386 371 −651 −1514 −1000 — 275 394 45 96 359 117 −369 −294 −249 — 3 −1816 −2377 −1532 −1832 −1492 −859 972 −254 2848 — 275 394 45 96 359 117 −369 −294 −249 — The amino acids are represented by the one letter code.

The first line for each position reports the match emission scores: probability for each amino acid to be in that state (highest score is highlighted). The second line reports the insert emission scores, and the third line reports on state transition scores: M→M, M→I, M→D; I→M, I→I; D→M, D→D; B→M; M→E. Table 7 shows that in the Group I profile HMM, methionine has a 4620 probability of being in the first position, the highest probability.

Step 4. Test the Specificity and Sensitivity of the Built Profile HMM

The Group I profile HMM was evaluated using hmmsearch, with the Z parameter set to 1 billion, for the ability to discriminate Group I members from those of Group II. The hmmsearch program takes the hmm file for the Group I profile HMM and all the sequences from both groups and assigns an E-value score to each sequence. This E-value score is a measure of fit to the Profile HMM, with a lower score being a better fit. The resulting score assignment list is provided in the appendix as Table 2. The Profile HMM clearly distinguished Group I members from Group II members since there was a large margin of E-value difference between the worst scoring Group I member (2.2e-181) and the best scoring Group II member (1.5e-07).

This analysis shows that the Profile HMM prepared for Group I XI proteins distinguishes Group I from Group II XI proteins. The Group I Profile HMM provides a structure that is linked to XI proteins that are functionally similar to XI of A. missouriensis.

Example 5 Construction of Chimeric P_(gapS)-xylA Genes for Group 1 XIs and Assembly of Double Crossover Suicide Vectors

The xylose isomerases from Geodermatophilus obscurus DSM 43160 (GOxylA; SEQ ID NO:64), Mycobacterium smegmatis str. MC2 155 (MSxylA; SEQ ID NO:10), Salinispora arenicola CNS-205 (SAxylA; SEQ ID NO:18), and Xylanimonas cellulosilytica DSM 15894 (XCxylA; SEQ ID NO:40) all belong to Group 1 xylose isomerases, based on amino acid sequence analysis described in Example 4. The sequences encoding these proteins were each optimized (SEQ ID NOs:335, 336, 337, and 338, respectively) for expression in Z. mobilis according to codon bias of Z. mobilis ZM4, and synthesized de novo as DNA fragments bounded by SpeI and XhoI sites, and with the coding region adjacent to a mutant Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene promoter (P_(gapS); SEQ ID NO:339) by GenScript Corporation (Piscataway, N.J.). The P_(gapS) is an improved P_(gap) that has a mutation which is a “G” to “T” change at position 116 of the natural promoter fragment (P_(gap)), that increases expression from the promoter, as disclosed in US 2009-0246876. The SpeI-P_(gapS)-xylA-XhoI fragments were cloned into pUC57 at the EcoRV site by GenScript Corporation (Piscataway, N.J.). The resultant intermediate plasmids were called pUC57-P_(gapS)GOxylA, pUC57-P_(gapS)MSxylA, pUC57-P_(gapS)SAxylA, and pUC57-P_(gapS)XCxylA. The optimized xylA coding sequences are GOxylA (SEQ ID NO:335), MSxylA (SEQ ID NO:336), SAxylA (SEQ ID NO:337), and XCxylA (SEQ ID NO:338).

Common molecular cloning methods were used to construct DCO suicide vectors. First, plasmid pARA356 (described in Example 1) was modified to add an XhoI site between the LBxylA and araD3′UTR sequences as follows. First, an araD3′UTR fragment was PCR amplified from pARA356 using forward primer ara368 (SEQ ID NO:345) and reverse primer ara97 (SEQ ID NO:319). The ara368 primer added SpeI and XhoI sites to the 5′ end of araD3′UTR, and ara97 added HindIII, FseI, and EcoRI sites to the 3′ end of araD3′UTR. The PCR product was digested with SpeI and EcoRI. The Pgap-LBxylA-araD3′UTR segment in pARA356 has a 5′ SpeI site and a 3′ EcoRI site. This segment was removed by digestion with SpeI and EcoRI and it was replaced by the above SpeI-XhoI-araD3′UTR-HindIII-FseI-HindIII-EcoRI PCR product. The resulting intermediate plasmid pARA356D has same sequence as pARA356, except P_(gap)-LBxylA was replaced by a XhoI site.

The four P_(gapS)-xylA fragments described above were isolated from the pUC57-based plasmids following SpeI and XhoI digestion, and cloned into the XhoI-modified pARA356D between the SpeI and XhoI sites to replace the P_(gap)-LBxylA fragment. The resulting four DCO suicide vectors were pARA356-GOxylA, pARA356-MSxylA, pARA356-SAxylA, and pARA356-XCxylA. These vectors are identical to pARA356 except that their chimeric xylA genes are expressed from P_(gapS).

As controls, AMxylA in pARA355 and ECxylA in pARA357 were used as representatives for Group I and II xylAs, respectively. However, since P_(gapS) was employed to express the four new Group 1 xylA genes, the P_(gap) promoters controlling AMxylA in pARA355 and ECxylA in pARA357 were changed to P_(gapS). For this purpose, a 319-bp P_(gapS) OLE-PCR fragment was synthesized from pARA356-XCxylA by PCR, using forward primer ara10 (SEQ ID NO:340) and reverse primer ara401 (SEQ ID NO:341); a 1,229-bp P_(gapS)-AMxylA OLE-PCR fragment was synthesized from pARA355 by PCR, using forward primer ara402 (SEQ ID NO:342) and reverse primer ara403 (SEQ ID NO:343); and a 1,367-bp P_(gapS)-ECxylA OLE-PCR fragment was synthesized from pARA357 by PCR, using forward primer ara402 and reverse primer ara404 (SEQ ID NO:344). One PCR reaction consisted of 50 μL AccuPrime Pfx SuperMix (Invitrogen, Carlsbad, Calif.), 1 μL of 40 ng/μL DNA template, and 1 μL of 10 μM forward and reverse primers. Reactions were carried out on an Eppendorf Mastercycler (Hemburg, Germany), following a hot start PCR program with 35 cycles of denaturing at 94° C. for 1 min/annealing at 56° C. for 1 min/extension at 72° C. for 2 min. The P_(gapS) OLE-PCR fragment included the entire P_(gapS), a 5′ SpeI site, and a 3′ start codon. The P_(gapS)-AMxylA and P_(gapS)-ECxylA OLE-PCR fragments contained an AMxylA and an ECxylA coding sequence, respectively. Each had a 36-nt 5′ sequence that matches the last 36 nt of P_(gapS) and a 3′ XhoI site. Furthermore, SpeI-P_(gapS)-AMxylA-XhoI and SpeI-P_(gapS)-ECxylA-XhoI fragments were synthesized by overlapping PCR (OLE-PCR). PCR reactions were set up as described above, but two templates were included. SpeI-P_(gapS)-AMxylA-XhoI was amplified from P_(gapS) and P_(gapS)-AMxylA OLE-PCR fragments by using forward primer ara10 and reverse primer ara403, while SpeI-P_(gapS)-ECxylA-XhoI was amplified from P_(gapS) and P_(gapS)-ECxylA OLE-PCR fragments by using forward primer ara10 and reverse primer ara404. Both SpeI-P_(gapS)-AMxylA-XhoI and SpeI-P_(gapS)-ECxylA-XI were digested with SpeI and XhoI, subjected to agarose gel electrophoresis, and purified by using QIAquick Gel Purification Kit (Qiagen). The DNA fragments were cloned into modified pARA356 (described above) between SpeI and XhoI sites to replace the P_(gap)-LBxylA fragment. The resulting two DCO suicide vectors were called pARA356-AMxylA and pARA356-ECxylA. All vectors were propagated in DH5α E. coli cells and prepared by using a QIAprep Spin Miniprep Kit.

Example 6 Integration of Chimeric P_(gapS)-xylA Genes into ZW641 and Characterization of Their Expression

This Example describes integration and expression of P_(gapS)-xylA chimeric genes in strain ZW641, described in Example 2, and demonstrates that the four tested Group I XIs indeed function better than Group II XIs in Z. mobilis.

Competent cells of strain ZW641-1A were prepared and transformed separately with pARA356-AMxylA, pARA356-ECxylA, pARA356-GOxylA, pARA356-MSxylA, pARA356-SAxylA, or pARA356-XCxylA. Transformants were selected on MMG5-Spec250 plates and analyzed by PCR for integration of the introduced P_(gapS)-xylA genes as described previously (see Example 2). The resultant strains were named ZW641-ara356-AMxylA, ZW641-ara356-ECxylA, ZW641-ara356-GOxylA, ZW641-ara356-MSxylA, ZW641-ara356-ASxylA, and ZW641-ara356-XCxylA strains. Among these strains, ZW641-ara356-AMxylA and ZW641-ara356-ECxylA were made as control strains since Example 3 demonstrated that the AMxylA Group 1 xylose isomerase was highly active in Z. mobilis, while ECxylA was the better enzyme of two tested Group II xylose isomerases.

To examine the growth of these six new strains in xylose and compare them with the parental strain ZW641-1A, all strains were subjected to a 96-hour shake flask fermentation in xylose. In the assay, each strain was grown overnight in 3 mL MRM3G5 at 30° C. with 150 rpm shaking. Cells were harvested, washed with MRM3×10 (same as MRM3G5 but 50 g/L glucose was replaced with 100 g/L xylose), and resuspended in MRM3×10 to OD₆₀₀ of about 0.1. Twenty-five milliliters of the suspension were placed in a 50 mL screw capped VWR centrifuge tube and grown at 30° C. with 150 rpm shaking for a 96-hour time course. During the time course, OD₆₀₀ was measured at 0, 24, 48, 72, and 96 hours. The resulting growth curve is shown in FIG. 6. It shows that similar to ZW1-ara355 and ZW1-ara357 strains analyzed in Example 3, ZW641-ara356-AMxylA grew to a cell density approximately three times higher than ZW641-ara356-ECxylA at end of the fermentation. ZW641-ara356-AMxylA had an OD₆₀₀ of 3.43, while ZW641-ara356-ECxylA reached 1.18. Both strains grew faster than ZW641. The other four strains all grew faster than ZW641-ara356-ECxylA. Their cell densities at end of the fermentation were between those of ZW641-ara356-AMxylA and ZW641-ara356-ECxylA.

To measure the metabolic profile of each strain, a 1 mL sample of each culture was collected at the 72-hour point. The samples were centrifuged at 10,000× g to remove cells. The supernatant was filtered through a 0.22 μm Costar Spin-X Centrifuge Tube Filter and analyzed by running through a BioRad Aminex HPX-A7H ion exclusion column with 0.01 NH₂SO₄ at a speed of 0.6 mL/min at 55° C. on an Agilent 1100 HPLC system to determine ethanol and xylose concentrations. The results given in Table 8 show that faster growth correlated with higher xylose utilization and more ethanol production. These results suggest that the difference in growth is due to the difference in XI activity. All strains had better growth, ethanol production and xylose utilization than the ZW641 control, and all strains with Group 1 XIs performed better than the strain with the Group II E. coli XI.

TABLE 8 Cell growth, xylose consumption, and ethanol production after 72 hours culturing at 30° C. in MRM3X10. Growth Strain (OD₆₀₀) Ethanol (g/L) Xylose* (g/L) ZW641 0.22 0.0 96.1 ZW641-ara356-AMxylA 3.21 26.3 41.5 ZW641-ara356-ECxylA 0.59 1.6 92.9 ZW641-ara356-GOxylA 1.14 3.0 90.0 ZW641-ara356-MSxylA 1.75 8.4 78.6 ZW641-ara356-SAxylA 1.51 5.0 86.3 ZW641-ara356-XCxylA 2.80 19.4 56.1 MRM3X10⁺ na^(#) 0.0 96.1 *xylose remaining in the medium ⁺starting medium ^(#)na: not applicable

To further confirm that these six strains each had an XI activity level corresponding to their growth and metabolic profiles, protein extracts were prepared as assayed for protein concentration and xylose isomerase activity as in Example 3. Specific activity was calculated as unit per milligram of protein with background OD₅₄₀ of D-xylose subtracted from the original OD₅₄₀ reading prior to calculation of activity. Each assay was repeated 3 times. FIG. 7 shows the resulting average specific XI activities in each protein extract. The absolute specific activities are lower than those in Example 3, most likely due to different reaction conditions. However, comparison of relative specific activities clearly shows that the XI activities correspond to the growth rates of the strains. Faster growth is supported by higher XI activity, with the AMxylA having highest activity and ECsyIA the lowest activity. All of the Group I XIs have higher average activities than the Group II ECxylA.

TABLE 1 HMMER2.0 [2.2g]¹ NAME seed-short² LENG 397³ ALPH Amino⁴ MAP yes⁵ NSEQ 21⁶ DATE Mon Oct 19 14:08:50 2009⁷ XT −8455 −4 −1000 −1000 −8455 −4 −8455 −4 NULT −4 −8455⁸ NULE 595 −1588 85 338 −294 453 −1158 197 249 902 −1085 −142 −21 −313 45 531 201 384 −1998 −644⁹ HMM A C D E F G H I K L M N P Q R S T V W Y m->m m->i m->d i->m i->i d->m d->d b->m m->e −462 * −1868 1(M) −1476 −1441 −2702 −2503 −679 −2424 −1861 −88 −2024 240

−2293 −2670 −1975 −1997 −1977 −1568 −381 −1705 −1336 1 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −24 −6482 −7525 −894 −1115 −701 −1378 −462 * 2(M) 421 −976 −556 −40 −1303 −1252 −182 −920 65 −1119

1057 −1439 138 −354 1386 371 −651 −1514 −1000 2 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 3(Y) −926 −719 −2839 −2316 1366 −2394 −664 122 −1975 1177 430 −1816 −2377 −1532 −1832 −1492 −859 972 −254

3 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 4(Q) −587 −1894 98 1129 −2216 −1318 −135 −1916 288 −1916 −1086 6 −1535

−133 −466 476 −1535 −2124 −1485 4 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 5(P) 963 −839 −1538 −1576 −2513 −1059 −1614 −2104 −1628 −2420 −1708 −1177

−1478 −1771 −442 −597 −1456 −2726 −2418 5 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 6(T) −582 −1338 −802 −473 −2030 −1419 −515 −1548 1250 −1750 −1035 −569 −1741 −184 −76 −652

−1225 −2043 −1605 6 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 7(P) −453 −1271 −792 −423 −2113 −1308 −480 −1789 138 −1875 −1095 −508

−134 1323 87 −556 −1368 −2082 −1622 7 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 8(E) 540 −2335 997

−2648 −1254 −347 −2412 −241 −2400 −1594 99 −1622 34 −857 −639 −843 −1973 −2617 −1835 8 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 9(D) −1212 −2696

333 −2575 −1370 2184 −2849 −658 −2784 −2085 −32 −1828 −282 −1310 −968 −1255 −2414 −2702 −1831 9 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 10(R) −1360 −2277 −1623 −673 −2899 −2052 1299 −2378 2306 −2139 −1406 −717 −2040 268

−1247 −1137 −2097 −2071 −1820 10 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 11(F) −2605 −2164 −3410 −3518

−3034 −940 −1504 −3300 −1072 −1142 −2698 −3246 −2697 −2999 −2815 −2670 −1743 −274 806 11 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 12(T) −29 −659 −1587 −1468 −2300 −927 −1414 −1919 −1399 −2228 −1440 −1037 −1583 −1249 −1574 1473

−1259 −2551 −2190 12 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 13(F) −2605 −2164 −3410 −3518

−3034 −940 −1504 −3300 −1072 −1142 −2698 −3246 −2697 −2999 −2815 −2670 −1743 −274 806 13 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 14(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 14 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 15(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 15 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 16(W) −3232 −2594 −3508 −3648 −882 −2917 −1886 −3060 −3237 −2665 −2615 −3229 −3282 −3161 −2996 −3457 −3341 −3103

−495 16 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 17(T) −665 −1148 −2040 −2098 −2442 −1424 −1936 −1850 −1956 −2305 −1815 −1645 −2071 −1911 −2001 −909

−1444 −2672 −2426 17 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 18(V) −1365 −986 −3741 −3350 −1294 −3426 −2948 1806 −3144 −231 −144 −3097 −3349 −2976 −3194 −2702 −1365

−2628 −2205 18 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 19(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 19 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 20(W) −1622 −1592 −2164 −1843 1022 −2585 −106 −1330 −1522 −1235 −864 1189 −2631 −1288 −1607 −1710 −1559 −1277

2199 20 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 21(Q) −321 −1138 −459 136 −1344 −1359 −136 −875 203 −1115 −365 −201 −1496

−180 368 1172 505 −1531 −1013 21 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 22(G) 1024 −757 −1652 −1777 −2766

−1806 −2449 −1965 −2750 −1944 −1230 −1687 −1701 −2063 −356 −543 −1600 −2942 −2694 22 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 23(R) 1048 −1275 −1018 −415 −1643 −1529 −292 −1159 404 −641 −643 −522 −1703 44

−628 −541 −930 −1713 −1274 23 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 24(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 24 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 25(P) 963 −839 −1538 −1576 −2513 −1059 −1614 −2104 −1628 −2420 −1708 −1177

−1478 −1771 −442 −597 −1456 −2726 −2418 25 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 26(F) −2605 −2164 −3410 −3518

−3034 −940 −1504 −3300 −1072 −1142 −2698 −3246 −2697 −2999 −2815 −2670 −1743 −274 806 26 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 27(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 27 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 28(D) −597 −1663

181 −2185 187 −451 −1754 −339 −1983 −1221 −104 −1612 −106 −878 −546 −665 506 −2311 −1660 28 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 29(A)

−720 −1681 −1630 −2405 −986 −1571 −1985 −1615 −2310 −1559 −1157 760 −1436 −1759 −333 −477 −1332 −2646 −2336 29 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 30(T) −341 −685 −2089 −1804 −1436 −1424 −1480 −3 −1550 −954 −534 −1410 −1930 −1436 −1668 −700

1255 −2050 −1664 30 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 31(R) −2176 −2415 −2504 −1923 −3054 −2403 −1269 −3053 −167 −2856 −2350 −1844 −2662 −997

−2228 −2131 −2807 −2501 −2452 31 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 32(E) −248 −1658 −85

−1958 −810 119 −1690 834 −1663 −757 139 985 778 456 381 316 −1270 −1853 −1189 32 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 33(A)

−1507 557 375 −1789 −501 65 −1489 442 −1529 −658 597 −1340 492 860 −168 −194 −459 −1775 −1142 33 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 34(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 34 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 35(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 35 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 36(P) −441 −915 −1763 −1646 −1764 −1345 −1491 −927 −1474 −1451 −1021 −1327

−1412 −1607 −703 −724 708 −2237 −1825 36 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 37(V) 335 −792 −3174 −2729 −1174 −2763 −2203 1517 −2499 −357 −111 −2444 −2850 −2311 −2556 −1972 −1037

−2170 −1771 37 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 38(E) −909 −1965 24

  −1246 −1505 −347 −1841 −213 −1874 −1207 −228 −1785 −120 −648 −805 −890 −1573 −1601 1568 38 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 39(A)

−630 −1276 −809 −1410 236 −731 −999 −672 −1275 −522 −711 −1520 −537 −976 877 1188 793 −1715 −1298 39 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 40(V) −529 −777 −2238 −2105 −1525 −7 −1733 111 −1947 −1096 −672 −1661 −2112 −1793 −1998 −945 −758

−2136 −1758 40 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 41(H) −582 −1877 −351 773 −2233 −1449

−1912 686 −1839 −988 −97 −1545 1646 1256 −467 −492 −1531 −1962 −1406 41 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 42(R) −1587 −2371 −1979 −913 −3101 −2190 −224 −2514 2541 −2236 −1542 −885 −2171 194

−1477 −1330 −2260 −2127 −1955 42 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 43(L) −1974 −1575 −4060 −3545 −282 −3812 −2663 1310 −3204

857 −3408 −3401 −2583 −3003 −3063 −1891 178 −1830 −1733 43 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 44(A)

−604 −1805 −1733 −2482 −871 −1613 −2129 −1700 −2432 −1611 −1136 −1569 −1478 −1829 1682 −361 −1353 −2728 −2414 44 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 45(E) −1003 −2473 512

−2788 −831 −430 −2578 −247 −2533 −1768 24 −1719 707 −767 −793 −1016 −2149 −2707 −1955 45 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 46(L) −1959 −1558 −4063 −3546 −291 −3808 −2666 1414 −3212

850 −3406 −3399 −2589 −3010 −3056 −1876 210 −1837 −1742 46 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 47(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 47 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 48(A)

−638 −2146 −1978 −1831 −1146 −1655 −629 −1782 −1443 −953 −1374 −1775 −1605 −1861 −461 −489 571 −2360 −2006 48 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 49(Y) −2410 −2105 −2586 −2487 1214 −3046 2815 −1980 −2072 −1592 −1442 −1863 −3075 −1758 −2026 −2294 −2336 −2000 511

49 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 50(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 50 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 51(V) −1387 −977 −3821 −3371 −1118 −3516 −2833 2100 −3172 597 34 −3124 −3343 −2908 −3170 −2752 −1359

−2430 −2081 51 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 52(T) −316 −1039 −679 −633 −2149 −1120 −926 −1803 −741 −2069 −1321 1353 −1653 −671 −1045 −444

−1311 −2344 −1843 52 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 53(F) −2065 −1675 −3615 −3327

−3380 −1003 −121 −2974 1209 326 −2658 −3213 −2303 −2729 −2622 −1996 −549 −310 671 53 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 54(H) −2323 −2419 −1868 −1956 −1368 −2396

−3143 −1645 −2924 −2595 −1989 −2806 −1873 −1712 −2381 −2439 −2923 −1719 −395 54 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 55(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 55 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 56(D) −1205 −3003

1455 −3203 −1271 −537 −3082 −663 −2979 −2251 1491 −1755 −188 −1433 −903 −1247 −2583 −3173 −2230 56 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 57(D) −1425 −3134

881 −3374 −1343 −726 −3296 −972 −3221 −2585 25 −1881 −410 −1787 −1106 −1504 −2808 −3355 −2435 57 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 58(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 58 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 59(I) −584 −415 −2576 −2004 811 −2186 −1087

−1704 −44 421 −179 −2212 −1418 −1667 −1280 −535 1582 −1034 −637 59 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 60(P) 831 −858 −1530 −1581 −2527 −1073 −1627 −2121 −1640 −2436 −1732 −1189

−1494 −1780 −462 −619 −1475 −2736 −2431 60 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 61(F) −1404 −1445 −2265 −1857

−2371 −514 −956 −1089 −826 −548 −1590 −2505 −1217 1154 −1633 −1384 −986 −276 725 61 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 62(G) −1147 −2145 1555 −151 −3123

−1064 −3044 −1224 −3073 −2375 −443 −1969 −787 −1831 −1065 −1335 −2467 −3051 −2494 62 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 63(S) 1540 −619 −1644 −1374 −2259 −896 −1302 −1897 −1273 −2159 −1334 −974 −1528 −1100 −1502

1259 −1223 −2486 −2129 63 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 64(T) −278 −1411 820 210 −1980 −1166 −168 −1685 139 −1746 −888 −45 610 235 −352 1310

−1258 −1999 −1378 64 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 65(D) 775 −2108

884 −2418 −1237 −217 −2176 −19 −2160 −1317 112 719 185 −599 −488 −639 −1745 −2375 −1635 65 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 66(Q) 1223 −1288 −293 188 −1719 −1202 −83 −1395 258 −1490 −649 435 −1388

−204 588 1044 −1031 −1779 −1191 66 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 67(E) −786 −1958 234

−2471 −1312 −479 −2090 −260 −2221 −1480 −118 −1693 −120 −738 −684 705 −1709 −2489 −1828 67 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 68(R) −2176 −2415 −2504 −1923 −3054 −2403 −1269 −3053 −167 −2856 −2350 −1844 −2662 −997

  −2228 −2131 −2807 −2501 −2452 68 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −86 −4238 −7714 −942 −1061 −701 −1378 * * 69(E) 180 −1985 1618

−2269 −1227 −60 −2029 241 −1986 −1104 146 −1438 1147 −305 −338 −450 −1591 −2174 −880 71 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 70(Q) −93 −1588 −113 706 −1865 −138 790 −1583 816 −888 −690 129 −1311

26 758 459 −1186 −1798 −1145 72 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 71(H) −345 −877 −695 −213 −903 −685

1646 31 −776 −65 −380 −1577 906 −283 −494 −289 −359 −1209 −725 73 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 72(I) −367 −817 −3505 −3009 −998 −3128 −2307

−2771 715 78 −2719 −3035 −2503 −2747 −2310 −1131 2143 −2063 −1704 74 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 73(K) 361 −1699 596 415 −2015 −63 90 −1743

−1710 −809 113 −1347 537 931 −183 −235 −1322 −1892 −1235 75 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 74(R) −320 −1614 442 360 −1920 −41 31 −1626 443 −1643 −772 621 −1381 460

−229 −270 −563 −1865 −1229 76 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 75(F) −324 −1039 −2854 −2418

−2568 −1007 160 −2106 857 406 −2027 −2596 −1719 −2040 −1726 −1158 −29 −596 214 77 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 76(K) −650 −1908 −584 44 −2320 −1534 916 −1965

−1862 −1018 553 −1598 439 1858 2 −543 −1590 −1953 −1448 78 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 77(Q) 71 −1748 −305 251 −2095 −183 48 −1783 1408 −1737 −866 −20 −1452

774 −328 −355 −1392 −1894 −1306 79 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 78(A)

−723 −1876 −1947 −2635 −214 −1832 −2241 −1993 −2601 −1822 −1305 −1695 −1754 −2055 −360 −529 −1479 −2845 −2605 80 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 79(L) −1818 −1523 −3759 −3310 −316 −3460 −2478 536 −2926

784 −3124 −3244 −2443 −2783 −2733 −1782 616 −1795 −1623 81 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 80(D) 101 −1863

425 −2206 −1222 −98 −1948 669 −1938 −1069 103 −382 321 −339 397 −446 −1522 −2148 −1452 82 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 81(E) 1195 −2299 1673

  −2594 −1247 −290 −2366 −151 −2338 −1512 118 −1587 102 −761 −582 −772 −1923 −2546 −1769 83 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 82(T) −194 −791 −1643 −1654 −2474 22 −1622 −2066 −1652 −2413 −1662 −1202 −1715 −1503 −1776 −414

−1411 −2683 −2390 84 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 83(G) 439 −855 −1581 −1757 −2835

−1850 −2546 −2010 −2843 −2063 −1269 −1756 −1754 −2106 −457 −649 −1703 −2977 −2743 85 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 84(M) −740 −586 −2676 −2080 −191 −930 −1155 1108 −1758 1717

−1802 −2271 −1414 −1692 −1385 −677 372 −1016 −731 86 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 85(K) −131 −982 −588 −21 −1093 −1402 −93 359

−893 −147 −231 −1484 210 265 −362 838 279 −1327 −833 87 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 86(V) −521 −748 −233 −1105 −1131 −1713 −1055 404 −1134 −720 −220 −1100 −2000 −969 −1389 −914 300

−1701 −1269 88 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 87(P) −1095 −1645 −1418 −1291 −2556 −1705 −1272 −2442 −720 −2498 −1867 −1276

−1049 408 −1217 −1284 −2022 −2467 −2195 89 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 88(M) −953 −1122 −1705 −1356 −305 −2019 883 −460 −763 −333

−1283 −2194 −908 −858 −1254 −950 −521 −955 −237 90 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 89(V) 1289 −665 −1346 −207 −986 −1604 −837 217 −874 −666 −98 −970 −1860 −753 −1115 −757 −455

−1481 −1067 91 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 90(T) −194 −791 −1643 −1654 −2474 22 −1622 −2066 −1652 −2413 −1662 −1202 −1715 −1503 −1776 −414

−1411 −2683 −2390 92 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 91(T) 1048 −603 −1704 −1538 −2267 288 −1432 −1863 −1474 −2173 −1368 −1056 −1558 −1278 −1649 −205

−1204 −2525 −2195 93 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 92(N) −1161 −2498 1023 254 −2901 −1337 −713 −2897 −803 −2894 −2199

−1842 −402 −1449 −964 −1262 −2411 −2948 −2145 94 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 93(L) −1075 −1199 −2228 −2047 −923 −434 −1571 −385 −1753

  −140 −1841 −2396 −1672 −1788 −1474 −1191 −504 −1704 −1322 95 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 94(F) −1621 −1684 −10 −1676

−2340 −642 −1251 −1954 −1200 −952 −1552 −2602 −1572 −2091 −1778 −1662 −1250 −309 743 96 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 95(T) −248 −1135 326 −166 −2058 −1097 −537 −1731 −339 −1883 −1065 −315 −1509 −198 −783 1772

−1257 −2181 −1628 97 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 96(H) 265 −2079 1321 773 −2325 −1256

−2082 19 −2080 −1247 103 −1537 198 −538 −495 −631 −1679 −2300 −1577 98 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 97(P) −2089 −2190 −2567 −2787 −3372 −2253 −2663 −3607 −2895 −3590 −3215 −2612

−2846 −2839 −2316 −2418 −3135 −2975 −3234 99 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 98(V) 479 −552 −2113 −1863 −1375 −3 −1443 −29 −1694 −1045 −494 −1385 −1851 −1485 −1768 −608 −468

−1913 −1556 100 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 99(F) −2377 −1971 −2987 −2956

−3100 1295 −1675 −2615 −1307 −1196 −2083 −3140 −2054 −2431 −2384 −2334 −1754 478 1631 101 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 100(K) −186 −1836 −969 −558 −2577 −1685 −425 −2088

−2110 −1410 −652 −1925 −45 429 −978 −981 −1757 −2208 −1848 102 — −149 −498 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −86 −4238 −7714 −1698 −532 −701 −1378 * * 101(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 108 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 102(G) −920 −1508 −1339 −1295 −2751

−1377 −2630 −937 −2716 −2012 −1241 −2085 −1160 523 −1056 −1170 −2071 −2626 −2390 109 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −102 −6672 −4094 −894 −1115 −701 −1378 * * 103(G) 1678 −647 −1598 −1653 −2595

−1650 −2259 −1777 −2558 −1746 −1116 −1581 −1527 −1899 −241 −417 −1446 −2792 −2519 110 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −22 −6592 −7635 −894 −1115 −618 −1521 104(F) −2605 −2164 −3410 −3518

−3034 −940 −1504 −3300 −1072 −1142 −2698 −3246 −2697 −2999 −2815 −2670 −1743 −274 806 111 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 105(T) −665 −1148 −2040 −2098 −2442 −1424 −1936 −1850 −1956 −2305 −1815 −1645 −2071 −1911 −2001 −909

−1444 −2672 −2426 112 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 106(S) 2001 −616 −1754 −1668 −2497 −871 −1578 −2157 −1641 −2446 −1619 −1109 −1565 −1427 −1789

−361 −1370 −2732 −2410 113 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 107(N) −603 −1690 −182 42 −2219 −1348 −286 −1949 226 −1968 −1162

26 97 673 −546 −618 −1549 −2135 −1563 114 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 108(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 115 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 109(R) −1150 −1864 −1429 −882 −2591 −1829 −597 −2283 393 −2219 −1523 −923 1080 −238

−1166 −1141 −1945 −2211 −1924 116 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 110(W) 344 −1328 1619 227 −1582 −1248 −77 −1278 216 −1399 −584 −39 −1418 299 −257 1029 −265 −964

−1101 117 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 111(V) −1349 −988 −3690 −3306 −1278 −3360 −2895 1671 −3086 −226 −141 −3047 −3312 −2925 −3136 −2638 −1356

−2597 −2168 118 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 112(R) −2176 −2415 −2504 −1923 −3054 −2403 −1269 −3053 −167 −2856 −2350 −1844 −2662 −997

−2228 −2131 −2807 −2501 −2452 119 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 113(R) 1106 −1210 −925 −462 −2109 −352 −466 −1735 167 −1836 −1045 −526 −1629 −112

−468 −502 −1309 −2063 −1625 120 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 114(Y) −1833 −1522 −3003 −2757 2546 −2889 −152 −1096 −2433 −987 −712 −1944 −2891 −1848 −2268 −2038 −243 −1114 464

121 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 115(A)

−913 −1413 −1186 −2083 −1183 −1104 −1652 −747 −1929 −1234 −982 −1727 −890 214 −497 −570 −1201 −2274 −1896 122 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 116(I) −818 1001 −3111 −2515 678 −2491 −1402

−2173 1686 640 −2108 −2446 −1766 −2010 −1604 −758 1207 −1150 −857 123 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 117(R) 615 −1244 −985 −490 −2134 −1326 −441 −1752 263 −1841 −1057 −543 −1650 −82

238 −530 −1334 −2053 −1625 124 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 118(K) −1518 −2307 −1691 −846 −2975 −2112 −281 −2466

−2235 −1545 −856 −2148 126 1301 −1423 −1307 −2203 −2146 −1935 125 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 119(V) 334 −503 −1661 −1221 −1131 −1220 −917 −428 −1040 −959 −283 −984 −1644 −868 −1235 814 1722

−1545 −1167 126 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 120(I) −1801 −1400 −4009 −3498 −409 −3715 −2658

−3194 2239 733 −3319 −3361 −2625 −3020 −2951 −1730 515 −1907 −1779 127 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 121(R) −590 −1886 −363 992 −2256 −1091 1110 −1926 695 −1849 −997 −103 −1551 997

−475 −499 −1544 −1970 −1419 128 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 122(N) −525 −1683 10 202 −2130 −1278 −228 −1842 152 −1887 −1070

−1544 1246 −266 −453 843 −1452 −2112 −1492 129 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 123(M) −1469 −1138 −3630 −3124 −557 −3307 −2344 2828 −2803 593

−2896 −3124 −2415 −2714 −2510 −1422 784 −1868 −1641 130 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 124(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 131 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 125(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 132 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 126(A)

−677 −1768 −1791 −2628 1691 −1726 −2289 −1863 −2588 −1770 −1201 −1631 −1609 −1973 −280 −452 −1476 −2836 −2569 133 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 127(A)

−636 −2232 −1987 −1562 −1335 −1616 −72 −1780 −1097 −650 −1463 −1894 −1599 −1867 −627 −539 1482 −2167 −1804 134 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 128(E) −1877 −2708 −271

−3235 −1868 −1322 −3210 −1299 −3203 −2676 −849 −2342 −1076 −1736 −1723 −1961 −2857 −2976 −2640 135 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 129(L) −992 −910 −2475 −1914 −111 −2437 615 229 −1462

1583 −1768 −2392 −1292 −1486 −1534 −923 −34 −1023 −591 136 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 130(G) −302 −865 −1762 −1813 −2349

−1733 −1847 −1826 −2301 −1669 −1332 −1811 −1666 −1921 −535 −673 −35 −2637 −2293 137 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 131(A)

−995 −2154 −2261 −2604 −1267 −2060 −2035 −2233 −2502 −1934 −1631 −1961 −2058 −2240 −724 −871 −1499 −2817 −2619 138 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 132(K) −495 −1865 −2571564 −2231 −1381 33 −1927

−1845 −968 −21 −1480 1087 678 −101 −414 −1517 −1970 −1379 139 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 133(T) 15 −567 −2088 −1580 −827 −1880 −1130 1432 −1354 −387 83 −1415 −2081 −1179 −1499 −1015

1019 −1446 −1065 140 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 134(Y) −1352 −1117 −2812 −2425 2395 −2595 −217 −654 −2102 398 −289 −1774 −2598 −1607 −1976 −504 −1284 −635 320

141 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 118 −369 −294 −249 — −86 −4238 −7714 −565 −1626 −701 −1378 135(V) −879 1157 −3282 −2943 −1304 −2350 −2295 1015 −2647 −535 −296 −2399 −2667 −2450 −2627 −1648 −1002

−2267 −1857 143 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 136(M) 1321 −485 −2344 −1767 −356 −2101 −1016 611 −1491 715

−1554 −2136 −1223 −1516 −1184 −533 1286 −1047 −714 144 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 137(W) −3232 −2594 −3508 −3648 −882 −2917 −1886 −3060 −3237 −2665 −2615 −3229 −3282 −3161 −2996 −3457 −3341 −3103

−495 145 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 138(G) −629 −1215 −1479 −1608 −2971

  −1817 −2847 −1898 −3004 −2246 −1366 1410 −1693 −2073 −816 −993 −2058 −2924 −2780 146 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 139(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 147 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 140(R) −2176 −2415 −2504 −1923 −3054 −2403 −1269 −3053 −167 −2856 −2350 −1844 −2662 −997

−2228 −2131 −2807 −2501 −2452 148 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 141(E) −1877 −2708 −271

−3235 −1868 −1322 −3210 −1299 −3203 −2676 −849 −2342 −1076 −1736 −1723 −1961 −2857 −2976 −2640 149 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 142(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 150 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 143(A)

−600 −1841 −1807 −2491 −872 −1666 −2124 −1780 −2447 −1635 −1169 −1579 −1551 −1883 1108 −372 −1350 −2748 −2442 151 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 144(E) −1877 −2708 −271

−3235 −1868 −1322 −3210 −1299 −3203 −2676 −849 −2342 −1076 −1736 −1723 −1961 −2857 −2976 −2640 152 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 145(Y) −366 −612 −1417 −941 −254 −1575 −466 −250 −742 −584 46 −879 −1770 −596 −952 1383 −382 905 −768

153 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 146(D) −1105 −2793

1424 −3056 1647 −501 −2904 −567 −2827 −2074 116 −1726 −144 −1292 −838 −1143 −2419 −3024 −2136 154 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 147(A)

−621 −1648 −1495 −2508 1640 −1463 −2219 −1491 −2440 −1580 −1023 −1533 −1271 −1696 1504 −328 −1400 −2709 −2377 155 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 148(A)

  −588 −1757 −1515 −2231 −893 −1384 −1850 −1406 −2137 −1324 −1039 −1540 −1216 −1597 1059 1273 −1189 −2483 −2146 156 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 149(K) −1004 −1906 −751 −464 −2680 668 −524 −2356

−2305 −1553 −618 −1920 −150 132 −968 −1013 −1956 −2316 −1940 157 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 150(D) −1145 −2665

357 −2959 −1353 −536 −2796 1099 −2743 −2006 −4 −1786 −182 −1024 −906 −1172 −2352 −2877 −2097 158 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 151(V) 556 −703 −2845 −2356 −868 −2476 −1724 1032 −2090 672 119 −2092 −2570 −1874 −2129 −1644 −865

−1729 −1361 159 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 152(R) 307 −1186 −699 −235 −2036 −283 −362 −1698 145 −1783 −956 −359 −1520 12

677 −375 −1252 −2024 −1524 160 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 153(D) 966 −1545

387 −1839 −201 57 −1545 1069 −1575 −700 91 −1343 486 −66 −11 −210 −337 −1814 −1172 161 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 154(A)

−635 −2197 −1970 −1633 −1272 −1616 −227 −1767 −1190 −730 −1428 −1852 −1587 −1854 −571 −520 1219 −2215 −1854 162 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 155(W) −1671 −1400 −3325 −2868 233 −3039 −1268 0 −2363 2540 532 −2472 −2926 −1994 −2227 −2277 −1599 −418

45 163 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 156(D) −1386 −3150

1221 −3366 −1320 −681 −3279 −911 −3191 −2536 63 −1851 −357 −1729 −1063 −1456 −2785 −3348 −2403 164 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 157(R) −1492 −1910 −2040 −1238 −1130 −2207 −499 −1858 364 −1773 −1220 −1192 −2291 −345

  −1537 −1364 −1712 2966 −659 165 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 158(M) −671 −553 −2451 −1885 −21 −2161 −868 387 −1554 249

−1603 −2178 −1263 −1528 −1248 −615 1153 −731 1912 166 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 159(R) −1586 −2370 −1977 −912 −3100 −2189 −223 −2513 2555 −2235 −1541 −885 −2170 194

−1476 −1329 −2260 −2127 −1954 167 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 160(E) −1877 −2708 −271

−3235 −1868 −1322 −3210 −1299 −3203 −2676 −849 −2342 −1076 −1736 −1723 −1961 −2857 −2976 −2640 168 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 161(A)

−659 −1524 −1392 −2402 874 −1398 −2082 −1414 −2320 −1489 −994 377 −1210 −1630 −211 −359 −1347 −2615 −2271 169 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 162(F) −1311 −1025 −3449 −2891

−2994 −1698 606 −2578 1623 733 −2548 −2836 −2068 −2410 −2134 −1242 1636 −1220 −793 170 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 163(D) −1226 −2864

438 −3143 −1293 −620 −3082 −772 −3012 −2309 2528 −1798 −288 −1527 −951 −1296 −2581 −3152 −2243 171 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 164(L) −982 −834 −2810 −2255 1392 −2500 −1292 389 −1949

736 −1995 −2463 −1577 −1892 −1612 726 161 −1047 −640 172 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 165(M) 438 −905 −2819 −2258 −264 −2565 −1490 453 −1943 2160

−2061 −2520 −1607 −1918 −1683 −989 202 −1306 −1076 173 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 166(G) 2232 −685 −1742 −1778 −2655

−1733 −2323 −1870 −2618 −1798 −1198 −1634 −1614 −1983 −284 −460 −1498 −2857 −2591 174 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 167(E) 823 −1941 898

−2233 −330 −41 −1990 272 −1949 −1063 145 −1423 1246 −270 −312 −417 −1552 −2138 −1425 175 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 168(Y) −2742 −2290 −3014 −3139 701 −3008 −615 −2258 −2836 −1879 −1835 −2399 −3220 −2421 −2640 −2733 −2781 −2314 18

176 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 169(V) 631 −431 −2194 −1660 −618 −1885 −1021 1372 −1413 −302 239 −1431 −2038 −1190 −1486 234 −449

−1192 −819 177 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 170(T) −500 −1770 −32 1487 −2088 −1320 −71 −1768 1131 −1785 −944 −2 −1493 344 −18 −397

−1398 −1998 −1382 178 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 171(D) 437 −2189

985 −2514 −1237 −262 −2284 −99 −2259 −1424 112 −1561 134 −696 605 −707 −1841 −2470 −1711 179 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 172(Q) −1116 −2167 −1119 −394 −2714 −1859 −124 −2252 1946 −2066 −1298 −521 −1894

1231 −1001 −946 −1936 −2060 −1719 180 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 173(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 181 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 174(Y) −2742 −2290 −3014 −3139 701 −3008 −615 −2258 −2836 −1879 −1835 −2399 −3220 −2421 −2640 −2733 −2781 −2314 18

182 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 175(D) −827 −2247

406 −2712 1546 −431 −2510 −378 −2495 −1697 868 −1641 −63 −1011 −654 −878 −1542 −2708 −1921 183 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 176(L) −604 −460 −2538 −1944 −130 −2143 −956 1316 −1614

679 −1640 −2146 −1291 −1551 −1226 89 434 −843 1377 184 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 177(R) −1151 −2083 −1284 −523 −2658 −1893 −175 −2181 1390 −2032 −1286 −614 244 239

−1068 −991 −1886 −2044 −1735 185 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 178(F) −1690 −1368 −3472 −3107

−3159 −1180 1363 −2783 394 347 −2559 −3051 −2224 −2601 −2369 −1641 22 −559 349 186 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 179(A)

−995 −2154 −2261 −2604 −1267 −2060 −2035 −2233 −2502 −1934 −1631 −1961 −2058 −2240 −724 −871 −1499 −2817 −2619 187 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 180(I) −1681 −1285 −3919 −3432 −552 −3632 −2662

−3136 1690 578 −3234 −3343 −2665 −3012 −2872 −1625 767 −2003 −1807 188 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 181(E) −1877 −2708 −271

−3235 −1868 −1322 −3210 −1299 −3203 −2676 −849 −2342 −1076 −1736 −1723 −1961 −2857 −2976 −2640 189 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 182(P) −2089 −2190 −2567 −2787 −3372 −2253 −2663 −3607 −2895 −3590 −3215 −2612

−2846 −2839 −2316 −2418 −3135 −2975 −3234 190 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 183(K) −1892 −2397 −1701 −1300 −3050 −2216 −948 −2829

−2696 −2116 −1348 −2450 −619 78 −1865 −1815 −2562 −2460 −2321 191 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 184(P) −2089 −2190 −2567 −2787 −3372 −2253 −2663 −3607 −2895 −3590 −3215 −2612

−2846 −2839 −2316 −2418 −3135 −2975 −3234 192 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 185(N) −1468 −2069 −762 −997 −2634 −1768 −1516 −3059 −1508 −3127 −2580

−2312 −1391 −1789 −1503 −1700 −2572 −2658 −2191 193 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 186(E) −1877 −2708 −271

−3235 −1868 −1322 −3210 −1299 −3203 −2676 −849 −2342 −1076 −1736 −1723 −1961 −2857 −2976 −2640 194 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 187(P) −2089 −2190 −2567 −2787 −3372 −2253 −2663 −3607 −2895 −3590 −3215 −2612

−2846 −2839 −2316 −2418 −3135 −2975 −3234 195 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 188(R) −2176 −2415 −2504 −1923 −3054 −2403 −1269 −3053 −167 −2856 −2350 −1844 −2662 −997

−2228 −2131 −2807 −2501 −2452 196 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 189(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 197 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 190(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 198 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 191(I) −1608 −1307 −3432 −3238 −1179 −3095 −2687

−2941 −191 −206 −2987 −3216 −2818 −2915 −2635 −1653 832 −2363 −1952 199 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 192(Y) −1651 −1320 −3317 −2937 1876 −2994 −612 −279 −2586 2172 216 −2231 −2886 −1963 −2359 −2131 −1568 −559 4

200 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 193(L) −2071 −1671 −3975 −3513 1646 −3704 −1951 276 −3199

835 −3187 −3331 −2460 −2935 −2941 −1974 −311 −1161 −569 201 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 194(P) 1158 −816 −1549 −1568 −2492 −1042 −1594 −2082 −1610 −2398 −1676 −1163

−1455 −1757 −417 −570 −1432 −2710 −2399 202 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 195(T) −665 −1148 −2040 −2098 −2442 −1424 −1936 −1850 −1956 −2305 −1815 −1645 −2071 −1911 −2001 −909

−1444 −2672 −2426 203 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 196(V) 290 −736 −3157 −2642 −923 −2834 −1963 1476 −2393 482 102 −2384 −2806 −2152 −2407 −1989 −981

  −1855 −1485 204 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 197(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 205 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 198(H) −647 −1406 −537 −467 −1335 −1374

−1931 −374 −2000 −1310 −576 −1796 −456 −639 836 −788 −1532 −1683 −889 206 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 199(A)

−598 −1408 −1011 −1291 553 −847 −790 −855 −1123 1960 −850 −1597 −722 −1095 −357 −292 −511 −1660 −1263 207 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 200(L) −1959 −1558 −4063 −3546 −291 −3808 −2666 1414 −3212

850 −3406 −3399 −2589 −3010 −3056 −1876 210 −1837 −1742 208 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 201(A)

−995 −2154 −2261 −2604 −1267 −2060 −2035 −2233 −2502 −1934 −1631 −1961 −2058 −2240 −724 −871 −1499 −2817 −2619 209 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 202(F) −2151 −1753 −3530 −3327

−3318 −783 −347 −2962 686 53 −2548 −3220 −2300 −2714 −2589 −2099 −722 −95 953 210 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 203(I) −1413 −1022 −3801 −3407 −1219 −3492 −2985

−3199 −111 −62 −3161 −3383 −2998 −3231 −2772 −1408 1813 −2596 −2206 211 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 204(E) 90 −1897 443

−2185 −1230 1725 −1936 326 −1898 −1010 138 −1411 1242 −197 −292 −385 −1505 −2086 −1385 212 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 205(R) −351 −1687 −179 891 −1996 −826 66 −1700 567 −1680 −800 50 −1394 1328

−249 1128 −1307 −1867 −1245 213 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 206(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 214 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 207(E) −1261 −2925 1287

−3172 −1326 −611 −3046 −721 −2978 −2273 46 −1812 −271 −1450 −976 −1310 −2573 −3131 −2258 215 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 208(R) −1403 −2166 −1551 −797 −2257 −2051 2088 −2287 844 −2112 −1431 −830 −2113 68

−1333 −1230 −2033 −1908 −1480 216 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 209(P) −721 −1287 −1519 −1649 −2984 1028 −1849 −2874 −1919 −3027 −2289 −1426

−1736 −2085 −907 −1079 −2116 −2912 −2790 217 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 210(E) −1251 −2967 1669

−3197 −1308 −591 −3075 −714 −2993 −2283 76 −1796 −249 −1463 −958 −1299 −2592 −3159 −2259 218 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 211(L) −904 −844 −2403 −1784 −342 −2365 −1121 1250 −1186

628 −1672 −2331 −1162 772 −1456 −834 204 −1214 −915 219 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 212(Y) −1403 −1120 −3032 −2663 2477 −2694 −320 −428 −2317 −519 −196 −1924 −2681 −1767 −2127 −1818 −1336 1473 248

220 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 213(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 221 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 214(V) −1401 −1014 −3772 −3281 −840 −3424 −2588 1734 −3048 1458 291 −3027 −3233 −2685 −2984 −2633 −1359

−2135 −1864 222 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 215(N) −1468 −2069 −762 −997 −2634 −1768 −1516 −3059 −1508 −3127 −2580

−2312 −1391 −1789 −1503 −1700 −2572 −2658 −2191 223 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 216(P) −1085 −1645 −1400 −1250 −2540 −1703 −1221 −2412 −640 −2466 −1828 −1243

−987 539 −1201 −1262 −1999 −2445 −2166 224 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 217(E) −1877 −2708 −271

−3235 −1868 −1322 −3210 −1299 −3203 −2676 −849 −2342 −1076 −1736 −1723 −1961 −2857 −2976 −2640 225 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 218(V) −565 −483 −2432 −1897 1185 −2061 −1097 692 −1621 −87 343 −1617 −2167 −1361 −1634 −1181 1369

−1112 −695 226 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 219(G) 1216 −738 −1673 −1781 −2746

−1793 −2425 −1950 −2725 −1915 −1223 −1673 −1686 −2050 −337 −522 −1578 −2929 −2677 227 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 220(H) −2323 −2419 −1868 −1956 −1368 −2396

−3143 −1645 −2924 −2595 −1989 −2806 −1873 −1712 −2381 −2439 −2923 −1719 −935 228 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 221(E) −1877 −2708 −271

−3235 −1868 −1322 −3210 −1299 −3203 −2676 −849 −2342 −1076 −1736 −1723 −1961 −2857 −2976 −2640 229 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 222(Q) −600 −1482 −475 −254 −2078 −1374 −473 −1739 63 −1831 −1129 −420 −1705

−202 −619 958 −1394 −2115 −1591 230 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 223(M) −1736 −1678 −2975 −2821 −903 −2619 −2137 −342 −2336 24

−2581 −2886 −2281 −2280 −2241 −1843 −654 −1928 −1574 231 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 224(A)

−600 −1848 −1825 −2494 −873 −1680 −2122 −1800 −2451 −1642 −1177 −1583 −1569 −1896 972 −376 −1349 −2754 −2449 232 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 225(G) −596 −1353 −455 −651 −2765

−1238 −2694 −1243 −2817 −2057 1271 −1821 −1009 −1597 −689 −896 −1982 −2836 −2358 233 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 226(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 234 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 227(N) −1468 −2069 −762 −997 −2634 −1768 −1516 −3059 −1508 −3127 −2580

−2312 −1391 −1789 −1503 −1700 −2572 −2658 −2191 235 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 228(F) −2605 −2164 −3410 −3518

−3034 −940 −1504 −3300 −1072 −1142 −2698 −3246 −2697 −2999 −2815 −2670 −1743 −274 806 236 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 229(T) −156 −631 −1441 −1038 −1327 −1213 −890 −577 −867 −1099 −449 −889 1925 −751 −1111 −410

948 −1720 −1318 237 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 230(H) 299 −1594 −259 23 −1760 −1411

−1605 285 −1670 −904 −212 −1624 1240 −25 −563 −577 −1296 −1828 −1224 238 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 231(G) 1216 −738 −1673 −1781 −2746

−1793 −2425 −1950 −2725 −1915 −1223 −1673 −1686 −2050 −337 −522 −1578 −2929 −2677 239 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 232(I) −1412 −1014 −3818 −3423 −1244 −3514 −3011

−3223 −139 −82 −3177 −3397 −3025 −3258 −2792 −1405 1920 −2622 −2228 240 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 233(A)

−702 −1841 −1506 −1222 −1365 −1211 −382 −1249 −749 1643 −1220 −1832 −1146 −1400 −615 −514 −252 −1806 −1421 241 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 234(Q) −1762 −2287 −1132 −1144 −2520 −2040 −1289 −2773 −740 −2625 −2183 −1302 −2433

−881 −1765 −1840 −2519 −2457 −2076 242 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 235(A)

−995 −2154 −2261 −2604 −1267 −2060 −2035 −2233 −2502 −1934 −1631 −1961 −2058 −2240 −724 −871 −1499 −2817 −2619 243 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 236(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 244 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 237(W) −1283 −1528 932 −1130 736 −2224 −138 −1253 −1094 −1220 −765 −1084 −2321 −868 −1347 −1344 −1228 −1158

1985 245 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 238(A)

−1366 −313 34 −1635 −1335 1574 −1366 204 −1485 −707 −193 −1544 1074 −155 −433 −425 −1066 −1749 −1178 246 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 239(G) −831 −2052 172 754 −2493

−347 −2192 1024 −2191 −1409 −103 −1691 30 −296 −704 −829 −1804 −2375 −1762 247 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 240(K) −1892 −2397 −1701 −1300 −3050 −2216 −948 −2829

−2696 −2116 −1348 −2450 −619 78 −1865 −1815 −2562 −2460 −2321 248 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 241(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 249 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 242(F) −2151 −1753 −3530 −3327

−3318 −783 −347 −2962 686 53 −2548 −3220 −2300 −2714 −2589 −2099 −722 −95 953 250 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 243(H) −2323 −2419 −1868 −1956 −1368 −2396

−3143 −1645 −2924 −2595 −1989 −2806 −1873 −1712 −2381 −2439 −2923 −1719 −935 251 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 244(I) −1608 −1307 −3432 −3238 −1179 −3095 −2687

−2941 −191 −206 −2987 −3216 −2818 −2915 −2635 −1653 832 −2363 −1952 252 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 245(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 253 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 246(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 254 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 247(N) −1468 −2069 −762 −997 −2634 −1768 −1516 −3059 −1508 −3127 −2580

−2312 −1391 −1789 −1503 −1700 −2572 −2658 −2191 255 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 248(G) −1138 −1994 970 −393 −3114

−1239 −3051 −1406 −3111 −2421 −642 −2033 −992 −1944 −1114 −1363 −2451 −3025 −2576 256 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 249(Q) −1762 −2287 −1132 −1144 −2520 −2040 −1289 −2773 −740 −2625 −2183 −1302 −2433

−881 −1765 −1840 −2519 −2457 −2076 257 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 250(R) −494 −1670 −394 131 −1997 −1407 1541 −1693 597 −1689 −853 228 −1520 390

1217 −426 −1339 −1858 −1299 258 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 251(G) −454 −932 −1545 −1368 −1592

−1240 −1146 −1201 −1367 2191 −1171 −1868 −1149 −1371 −692 −687 −902 −2003 −1589 259 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 252(I) −252 −621 −1011 −601 −831 −1403 −480

−483 −684 1 362 344 −369 −758 147 −276 −145 −1217 −790 260 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 253(K) −1539 −2341 −1845 −864 −3043 −2150 −233 −2484

−2225 −1527 −857 −2149 183 1839 −1433 −1300 −2224 −2127 −1935 261 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 254(Y) −2984 −2211 −3668 −3802 2833 −3558 −44 −1957 −3405 −1449 −1443 −2318 −3461 −2386 −2945 −2790 −2877 −2089 685

262 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 255(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 263 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 256(Q) −1762 −2287 −1132 −1144 −2520 −2040 −1289 −2773 −740 −2625 −2183 −1302 −2433

−881 −1765 −1840 −2519 −2457 −2076 264 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 257(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 265 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 258(L) −1238 1377 −3271 −2822 −453 −2615 −1907 366 −2410

519 −2451 −2721 −2079 −2306 −1895 −1275 123 −1577 −1279 266 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 259(R) −435 −969 −1119 −575 −1319 −375 −421 −736 74 −1090 −402 −632 −1698 −167

−601 −453 1627 −1543 −1122 267 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 260(F) −1141 −1255 −2377 −2279

−2092 −569 −1041 −2100 −1019 −743 −1705 −2446 −1735 −2059 −76 −1293 −987 −121 942 268 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 261(G) −920 −1508 −1339 −1295 −2751

−1377 −2630 −937 −2716 −2012 −1241 −2085 −1160 523 −1056 −1170 −2071 −2626 −2390 269 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 262(H) 910 −1328 −303 212 −1640 −1258

−1308 356 −1405 −574 −44 −733 368 434 599 −227 −980 −1697 −1118 270 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −102 −6672 −4094 −894 −1115 −701 −1378 * * 263(G) −998 −2008 156 1372 −2814

−820 −2621 −763 −2678 −1963 −325 −1846 −517 −1251 −919 −1134 −2140 −2758 −2201 271 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −22 −6592 −7635 −894 −1115 −618 −1521 * * 264(D) −896 −2439

448 −2718 −1275 −358 −2532 −225 −2479 −1678 1424 −1648 21 249 −685 −902 −2082 −2659 −1868 272 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 265(L) −1226 −1270 −2434 −2156 −630 −2346 −1635 −84 −1796

285 −2000 −48 −1683 −1827 −1692 −1284 −360 −1630 −1298 273 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 266(R) 261 −1516 −309 264 −1768 −1323 57 −1437 1161 −420 −634 −7 −1406 1111

−261 663 −1102 −1724 −1141 274 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 267(A)

−968 −672 −395 −2180 −32 −670 −1885 −459 −2014 −1164 1678 −1481 −332 −871 1019 −328 −1306 −2284 −1769 275 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 268(A)

−694 −1839 −1891 −2627 246 −1791 −2258 −1948 −2591 −1793 −1261 −1663 −1699 −2026 −319 −489 −1473 −2840 −2589 276 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 269(F) −1633 −1375 −3311 −2975

−2991 −973 88 −2640 335 217 −2384 −2965 −2115 −2486 −2194 −1612 560 −373 594 277 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 270(W) −649 −554 −2266 −1731 1870 −2042 −407 −45 −1432 578 294 −1386 −2086 −1111 −1417 831 −590 6

661 278 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 271(L) −954 −821 −2749 −2189 −379 −2508 −1467 615 −1885

848 −1995 −2489 −1589 −1889 −1624 721 884 −1355 −1090 279 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 272(V) −1294 −1167 −3176 −3053 −1510 −2597 −2599 776 −2808 −665 −568 −2665 −2928 −2721 −2804 −2093 −1429

−2527 −2099 280 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 273(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 281 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 274(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 282 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 275(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 283 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 276(E) −1877 −2708 −271

−3235 −1868 −1322 −3210 −1299 −3203 −2676 −849 −2342 −1076 −1736 −1723 −1961 −2857 −2976 −2640 284 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 277(N) −343 −1453 −252 132 −2019 −1231 −165 −1718 264 −1761 −914

−1464 238 635 1541 −137 −1302 −1987 −1397 285 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 278(A)

−615 −1671 −1548 −2532 1980 −1508 −2242 −1558 −2469 −1610 −1047 −1538 −1327 −1748 1152 −334 −1411 −2738 −2415 286 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −1061 −6672 −969 −894 −1115 −701 −1378 * * 279(F) −62 −382 −890 −612

848 −159 −157 −517 −365 131 −522 −1382 −366 −722 −310 −160 −26 −381 402 287 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −43 −5654 −6697 −894 −1115 −1298 −753 * * 280(P) 53 −411 −960 −673 −945 −944 −591 −73 −495 −605 −126 −572

−444 −723 −198 −123 1167 −1421 −973 288 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −36 −5916 −6958 −894 −1115 −1541 −607 * * 281(N) −403 −1803 1732 803 −2196 600 10 −1981 99 −1980 −1180

−1229 381 −510 −235 −435 −1535 −2198 −1435 289 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −36 −5916 −6958 −894 −1115 −1541 −607 * * 282(G) −564 −931 −1093 −1247 −2240

−1339 −2241 −1464 −2403 −1833 −1104 −1599 −1344 −1556 −759 −890 −1685 −2020 −2051 290 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −36 −5916 −6958 −894 −1115 −1541 −607 * * 283(G) 1043 −280 −688 −700 −1858

−847 −1455 −840 −1779 −1026 −438 −1116 −671 −1053 164 18 −830 −2063 −1703 291 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −36 −5916 −6958 −894 −1115 −1541 −607 * * 284(P) −290 −1038 −466 −90 −1581 −1092 −104 −1283 527 −1372 −663 −208

220 1475 −341 −334 −968 −1564 −1136 292 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −36 −5916 −6958 −894 −1115 −349 −2218 * * 285(G) 638 −1161 −471 −124 −2022

−396 −1704 1009 −1808 −971 −280 −1477 −27 −463 540 −339 −1238 −2076 −1532 293 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 286(Y) −2887 −2205 −3496 −3544 1333 −3432 −88 −2037 −3047 −1553 −1514 −2272 −3386 −2288 −2715 −2719 −2795 −2134 1494

294 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 287(D) 192 −1858

1374 −2155 −1216 15 −1908 361 −1866 −969 151 −1383 1382 −169 −23 260 −1470 −2052 −1350 295 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 288(G) −1815 −2026 −2479 −2749 −3578

−2701 −3848 −3053 −3865 −3355 −2493 −2700 −2875 −2985 −2041 −2188 −3149 −3082 −3440 296 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 289(P) −2089 −2190 −2567 −2787 −3372 −2253 −2663 −3607 −2895 −3590 −3215 −2612

−2846 −2839 −2316 −2418 −3135 −2975 −3234 297 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 290(R) −1641 −2275 −1933 −1088 −2932 −2175 −424 −2500 1015 −2295 −1649 −1042 −2256 −38

−1586 −1455 −2254 −2181 −1996 298 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 291(H) −2323 −2419 −1868 −1956 −1368 −2396

−3143 −1645 −2924 −2595 −1989 −2806 −1873 −1712 −2381 −2439 −2923 −1719 −935 299 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 292(F) −2605 −2164 −3410 −3518

−3034 −940 −1504 −3300 −1072 −1142 −2698 −3246 −2697 −2999 −2815 −2670 −1743 −274 806 300 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 293(D) −2059 −2867

−532 −3452 −1877 −1516 −3665 −1847 −3616 −3145 −882 −2414 −1318 −2469 −1872 −2198 −3236 −3167 −2847 301 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 294(Y) 579 −1160 −2665 −2299 2734 −2531 −229 −710 −2012 −758 −361 −1718 −2573 −1556 −1937 −1652 −1288 −684 285

302 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 295(K) −1225 −2178 −1024 −471 −2433 −1882 2092 −2276

−2116 −1390 −596 −1974 157 713 −1121 −1075 −1982 −2013 −1586 303 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 296(P) 1158 −816 −1549 −1568 −2492 −1042 −1594 −2082 −1610 −2398 −1676 −1163

−1455 −1757 −417 −570 −1432 −2710 −2399 304 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 297(P) −197 −804 −966 −577 −1355 −1206 −599 −964 −444 231 −508 −598

−375 −760 1626 −323 −681 −1668 −1194 305 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 298(R) −2176 −2415 −2504 −1923 −3054 −2403 −1269 −3053 −167 −2856 −2350 −1844 −2662 −997

−2228 −2131 −2807 −2501 −2452 306 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 299(T) −665 −1148 −2040 −2098 −2442 −1424 −1936 −1850 −1956 −2305 −1815 −1645 −2071 −1911 −2001 −909

−1444 −2672 −2426 307 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 300(E) −1251 −2967 1669

−3197 −1308 −591 −3075 −714 −2993 −2283 76 −1796 −249 −1463 −958 −1299 −2592 −3159 −2259 308 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 301(D) −1120 −2685

438 −3120 658 −599 −2993 −733 −2934 −2207 768 −1759 −261 −1478 −875 −1202 −2475 −3126 −2235 309 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 302(F) −407 −383 −1904 346

−1828 −478 1038 −1078 161 475 −1148 −1876 −826 −1146 −877 −346 230 −488 1761 310 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 303(D) −981 −2590

1382 −2857 −1262 −422 −2656 −403 −2615 −1835 116 −1677 −53 −1074 −746 483 −2200 −2827 −1988 311 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 304(G) 439 −855 −1581 −1757 −2835

−1850 −2546 −2010 −2843 −2063 −1269 −1756 −1754 −2106 −457 −649 −1703 −2977 −2743 312 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 305(V) −1294 −1167 −3176 −3053 −1510 −2597 −2599 776 −2808 −665 −568 −2665 −2928 −2721 −2804 −2093 −1429

  −2527 −2099 313 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 306(W) −3232 −2594 −3508 −3648 −882 −2917 −1886 −3060 −3237 −2665 −2615 −3229 −3282 −3161 −2996 −3457 −3341 −3103

−495 314 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 307(A)

−1986 1345 1356 −2294 −1231 −146 −2041 102 −2030 −1173 114 −356 265 −456 −410 −533 −1618 −2244 −1527 315 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 308(S) −447 −881 −1613 −1364 1517 −1419 −806 −1058 −1261 −1223 −705 −1119 −1875 −1080 −1446

−648 −827 −983 −113 316 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 309(A)

−995 −2154 −2261 −2604 −1267 −2060 −2035 −2233 −2502 −1934 −1631 −1961 −2058 −2240 −724 −871 −1499 −2817 −2619 317 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 310(K) 1246 −1752 −354 687 −2095 −1400 24 −1767

−1730 −874 −63 −1490 459 1177 −382 −400 −1394 −1891 −1325 318 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 311(G) 1279 −1129 455 −229 −2280

−665 −1991 −519 −2113 −1277 −355 −1522 −330 −976 392 −436 −1424 −2385 −1830 319 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 312(C) −342

−1550 −1431 −1885 −1211 −1326 −1596 −1300 −1974 −1287 2515 −1793 −1235 −1463 −561 −633 −1160 −2187 −1732 320 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 313(M) −1538 −1273 −3473 −2967 −378 −3209 −2177 1411 −2583 795

−2776 −3045 −2211 −2502 −2413 −1492 381 −1708 −1493 321 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 314(R) −802 −1573 −1236 −713 −2404 −1580 −468 −2096 486 −2079 −1343 −728 −1867 −105

691 −850 −1698 −2130 −1757 322 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 315(M) −291 −866 −751 −323 −1102 −1363 −365 −625 −175 −903

1884 −1580 −129 −510 −445 862 −433 −1424 −950 323 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 316(Y) −2742 −2290 −3014 −3139 701 −3008 −615 −2258 −2836 −1879 −1835 −2399 −3220 −2421 −2640 −2733 −2781 −2314 18

324 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 317(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 325 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 318(I) −1718 −1320 −3950 −3454 −500 −3658 −2657

−3157 1891 635 −3261 −3346 −2650 −3014 −2895 −1657 685 −1967 −1796 326 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 319(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 327 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 320(K) −1892 −2397 −1701 −1300 −3050 −2216 −948 −2829

−2696 −2116 −1348 −2450 −619 78 −1865 −1815 −2562 −2460 −2321 328 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 321(E) −1027 −2709 1389

−2938 −1270 −422 −2771 −394 −2690 −1910 126 −1688 847 −1061 −772 −1042 −2301 −2880 −2022 329 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 322(R) −1262 −1929 −1576 −1005 −2676 −1901 −641 −2373 404 −2286 −1610 −1018 104 −286

−1282 −1246 −2043 −2243 −1991 330 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 323(A)

−817 −1288 −964 −1935 −1111 −906 −1465 −558 −1746 −1009 −813 −1616 −662 368 −363 −64 −1031 −2134 −1737 331 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 324(Q) 921 −1531 −150 983 −1783 −1243 90 −1481 984 −362 −643 89 −373

16 −170 −191 −1116 −1755 −1125 332 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 325(A)

−721 −1722 −1692 −2428 −988 −1617 −2000 −1677 −2335 −1590 −1188 277 −1495 −1805 −343 −489 −1343 −2672 −2372 333 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 326(F) −1141 −1255 −2377 −2279

−2092 −569 −1041 −2100 −1019 −743 −1705 −2446 −1735 −2059 −76 −1293 −987 −121 942 334 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 327(R) 26 −1636 −1533 −952 −2483 −1704 −566 −2043 472 −2068 −1404 −909 −1989 −212

−1021 −1002 −1707 −2177 −1875 335 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 328(A)

−1366 −122 1071 −2009 −1198 −396 −1654 −158 −1799 −1003 −190 −81 −44 −610 −402 −477 −1263 −2110 −1531 336 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 329(D) −1040 −2279

219 −2914 −1294 −744 −2643 −860 −2773 −2097 −113 −1811 −431 −1522 −889 170 −2183 −2988 −2215 337 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 330(P) −1078 −1646 −1388 −1222 −2530 −1702 −1185 −2393 −586 −2444 −1802 −1219

−944 631 −1190 −1247 −1984 −2429 −2146 338 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 331(E) −592 −2098 820

−2389 −1258 −116 −2150 202 −2093 −1225 119 −1495 1056 383 −427 −554 −1711 −2269 −1550 339 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 332(V) −864 1397 −3270 −2925 −1287 −2333 −2263 1008 −2628 −532 −284 −2379 −2649 −2425 −2605 −1626 −986

−2235 −1829 340 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 333(Q) −1116 −2159 −1088 −390 −2695 −1853 −132 −2237 1638 −2059 −1295 −519 −1896

1253 −1004 −949 −1924 −2060 −1716 341 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 334(E) 77 −1884 74

−2260 −1329 −186 −1946 248 −1959 −1140 −36 −1567 216 517 −508 −594 −1564 −2165 −1539 342 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 335(A)

−778 −1188 −678 −992 −1464 −510 −448 −265 351 −128 −707 −1690 −333 310 −572 −378 −302 −1373 −946 343 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 336(L) −273 −1147 −3152 −2607 −269 −2901 −1837 473 −2264

1696 −2426 −2787 −1885 −2206 −2051 −1280 127 −1497 −1323 344 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 337(A)

−1532 −474 87 −1918 −1382 −46 −1567 1215 −1601 −772 −145 −1504 365 1342 125 −380 −1223 −1818 −1283 345 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 338(A)

−972 −614 294 −1776 −1148 −611 −1301 −417 −1587 −829 −465 −1555 −323 −786 −354 962 −937 −1990 −1503 346 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 339(S) 1294 −832 −756 −278 −1210 −1235 −345 −856 −177 −1085 −331 −396 −201 −85 −555

−227 −590 −1486 1213 347 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 340(R) −570 −1686 −582 34 −1977 144 −15 −1637 1580 −1635 −817 −192 −157 4399

−500 −474 −1317 −1803 1086 348 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 46 96 359 117 −369 −294 −249 — −307 −2420 −7714 −266 −2570 −701 −1378 * * 341(V) −336 −353 −1746 340 −254 −1760 −562 286 −906 1013 531 −1061 −1812 −710 −1015 −805 −277

1260 −385 350 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 342(D) 352 −1357

274 527 −1272 −34 −1207 263 −1335 −525 −5 −1411 741 −215 −270 262 −916 −1662 −1069 351 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 343(E) −521 −1935 227

−2246 −1250 −100 −1987 223 −1965 −1102 97 607 1057 −287 155 −487 −1568 −2166 −1473 352 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 344(L) 546 −413 −2480 −1879 −125 −2068 −906 1031 −1548

714 −1574 −2081 −1226 −1484 −1149 −484 382 1382 −472 353 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 345(A)

−1490 227 381 −1734 4 86 −1428 735 −1476 −609 88 −377 512 391 −161 −178 −1072 −1730 −134 354 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 346(Q) −254 −1503 −183 1017 −1748 −1253 86 −1438 495 −297 −617 73 −1345

735 190 977 −1084 −1731 −1110 355 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 347(P) 421 −948 −865 −458 −1802 −1152 −548 −1416 −196 −1606 −823 −506

−237 384 −316 452 −1015 −1949 −1485 356 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 348(T) −276 −686 −1314 −885 −1018 −1380 −716 −410 −659 414 −163 −836 −135 −591 −897 −537

−256 −1462 −1045 357 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 349(L) 1151 −788 −2503 −1952 −325 −2317 −1281 427 −1656

1590 −1784 −2340 −1385 −1690 −1427 −817 257 −1248 −972 358 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 350(N) 1068 −1985 832 927 −2287 217 −111 −2043 157 −2016 −1147

−1465 308 −398 −378 −498 −1611 −2219 −1499 359 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 351(D) 515 −2116

1040 −2419 −1237 −204 −2180 1 −2157 −1311 118 1413 200 −577 −479 −630 −1746 −2368 −1626 360 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −214 −6672 −2965 −894 −1115 −701 −1378 * * 352(G) −1483 −1738 −2118 −2361 −3240

−2356 −3437 −2647 −3497 −2968 −2132 −2414 −2483 −2624 −1701 −1847 −2772 −2817 −3088 361 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −602 −6482 −1599 −894 −1115 −1010 −990 * * 353(E) −844 −1778 472

−2144 −1088 −364 −1914 −199 −2018 −1432 9 −1504 −76 −613 −726 −895 −1616 −2068 −1590 362 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −36 −5916 −6958 −894 −1115 −1541 −607 * * 354(S) 409 −277 −676 −434 −1615 472 −531 −1263 −415 −1493 −687 −281 −1062 −284 −715

1521 −699 −1824 −1402 363 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −36 −5916 −6958 −894 −1115 −349 −2218 * * 355(Y) 176 −326 −1853 −1280 −211 −1746 −587 276 −1009 1143 525 −1118 −1820 −790 −1087 −806 334 460 −736

364 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 356(Q) 1158 −1744 813 939 −2039 −1215 47 −1776 411 −1756 −861 138 −1359

−105 600 −85 −1356 −1956 −1275 365 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 357(D) 1036 −1772

823 −2060 −1224 1065 −1797 408 −1774 −882 135 −1370 477 479 −226 −297 −1379 −1971 −1290 366 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 358(L) −831 −865 −1889 480 −453 −2235 −1046 1057 −1158

523 −1421 −2241 −1015 −1320 −1316 −771 284 −1297 −968 367 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 359(L) −837 −924 −1941 −1334 −448 −2177 −874 129 765

1446 −1328 −2171 −793 −816 −1251 −762 −40 −1238 −923 368 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 360(A)

−1304 264 −12 −2084 −1143 −485 −1768 −315 −1902 −1096 676 −568 −145 −788 −383 −481 −1328 −2203 −1621 369 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 361(D) −1360 −3141

1479 −3350 −1311 −657 −3259 −874 −3165 −2499 78 −1836 −330 −1688 −1038 −1426 −2762 −3331 −2380 370 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 362(R) 1110 −1178 −914 −425 −2040 −1274 −431 −1667 185 −1776 −982 −495 −1601 −75

266 −459 −1250 −2016 −1566 371 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 363(S) 660 −658 −1132 −653 −1190 −1207 −583 −715 −506 248 −310 −639 −1546 −394 −817

985 −458 −1520 −1091 372 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 364(A)

−1109 −599 −215 −1913 −1164 −412 −1560 1070 −1701 −888 −346 −1504 −60 −397 906 −348 −1138 −1994 −1480 373 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 365(F) −1666 −1475 −2647 −2273

−2733 −126 −1109 −1832 −1025 −680 −1722 −2730 −1513 985 −1846 −1589 −1094 401 2740 374 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 366(E) 1231 −2217 1090

−2529 −1247 −283 −2286 −130 −2276 −1451 104 −1579 108 −726 −564 −741 −1853 −2493 −1734 375 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 367(D) −807 −2381

1701 −2662 −153 −299 −2456 −176 −2404 −1577 131 −1595 93 −801 −602 340 −1999 −2603 −1807 376 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 368(F) −2122 −1662 −3439 −3251

−3201 −250 −937 −2878 738 −472 −2219 −3110 −2117 −2574 −2364 −2033 −1127 427 2382 377 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 369(D) −106 −2081

302 −2719 −1222 −536 −2478 −516 −2518 −1742 −21 945 −185 −1136 −670 −895 −1991 −2748 −1995 378 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 370(V) 1420 −434 −1909 −1374 −548 −1743 −830 417 −1135 567 297 −1213 −880 −938 −1254 −846 −374

−1093 −720 379 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 371(D) −1069 −2768

2120 −3007 −1268 −462 −2850 −491 −2770 −2001 126 −238 −97 −1197 −805 −1095 −2370 −2962 −2084 380 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 372(A)

−1018 −521 219 −2069 −1094 −793 −1676 −652 −1914 −1144 −512 −1591 −518 −1012 194 −479 −1214 −2262 −1772 381 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −248 — −149 −3423 −7714 −422 −1979 −701 −1378 * * 373(A)

−605 −1051 −501 555 −1512 −336 −123 725 −487 179 −589 −1619 −212 −602 −527 −227 695 −1054 −619 383 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 374(A)

−1501 684 281 −1928 64 −79 −1628 270 −1677 −820 6 −1408 332 916 −249 −300 −1233 −1925 −1300 384 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 375(A)

−1589 −193 665 −1851 −1305 1059 −1534 504 −1565 −713 20 −1412 946 607 −274 −289 −1184 −1797 −1195 385 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 376(R) −1614 −2370 −2014 −948 −3100 −2204 −239 −2515 2045 −2241 −1554 −910 −2189 176

−1510 −1356 −2268 −2131 −1966 386 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −45 −6672 −5593 −894 −1115 −701 −1378 * * 377(G) −227 −995 −514 −621 −2598

−1104 −2383 −1085 −2534 −1712 700 −1597 −848 −1442 475 −540 −1636 −2744 −2250 387 — −149 −500 233 43 −381 399 106 −626 210 −466 −719 275 394 45 96 359 117 −369 −294 −249 — −1813 −3952 −620 −1278 −767 −674 −1422 * * 378(T) 788 −14 −411 −289 −946 −373 −321 3 −144 −602 −96 −129 −912 −138 −343 312

256 −1367 −928 391 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −380 −2195 −6258 −462 −1868 −273 −2536 * * 379(M) −645 −509 −2405 −1863 2203 −2065 −400 −15 −1534 −240

−1445 −2099 −1182 −142 −1141 −581 44 674 2154 393 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 380(A)

−667 −1488 −1343 −2399 1353 −1365 −2084 −1366 −2315 −1480 −970 504 −1166 −1595 −208 −355 −1350 −2606 −2254 394 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 381(F) −157 −954 −2834 −2359

−2528 −1072 238 −2050 1087 505 −2003 −2540 −1667 −1985 −1670 −1066 47 −705 20 395 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 382(E) −328 −925 −616

−1062 −545 −254 284 −67 −820 −134 −349 −1576 14 −460 −466 −286 1263 −1383 −892 396 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 383(R) −570 −1882 277 160 −2259 −1451 1440 −1932 1247 −1846 −988 −96 −1537 644

−453 −477 −1542 −1960 −1408 397 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 384(L) −1018 −1162 −2165 −1980 −936 −207 −1526 −403 −1702

−157 −1778 −2350 −1618 −1747 −1407 −1137 −503 −1693 −1309 398 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 385(D) −977 −2554

464 −2883 −1256 −450 −2718 −443 −2663 −1882 2380 −2 −85 −1116 −749 −1012 −2239 −2855 −2014 399 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 386(Q) −923 −1831 −422 −324 −2362 −1528 −632 −2219 −101 −2202 −1511 −528 80

−359 −896 −984 −1846 −2304 −1794 400 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 387(L) −124 −1187 −2771 −2463 −539 −2463 −1870 292 −2101

475 −2214 −2644 −1894 −2088 −1750 −1260 31 −1713 −1435 401 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 388(A)

−694 −1839 −1891 −2627 246 −1791 −2258 −1948 −2591 −1793 −1261 −1663 −1699 −2026 −319 −489 −1473 −2840 −2589 402 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 389(M) −586 −422 −2607 −2005 −236 −2162 −1033 1738 −1674 727

−1686 −2164 −1351 −269 −1247 −526 1278 −937 −619 403 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 390(E) −86 −2659 2289

−2918 −1261 −428 −2735 −423 −2675 −1894 125 −1682 −59 −1107 −758 −1026 −2265 −2876 −2019 404 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 391(H) −1063 −1596 −1226 −723 125 −1986

−1400 −6 −1381 −808 −779 −2060 −275 584 −1076 −975 −1242 −421 1920 405 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 392(L) −2228 −1908 −3555 −3373 −604 −3255 −2515 −62 −2963

358 −3207 −3275 −2644 −2802 −2988 −2232 −567 −1904 −1643 406 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 393(L) −1311 −1120 −3138 −2575 −246 −2891 −1784 463 −2223

1961 −2395 −2763 −1847 −2162 −2028 −31 113 −1452 −1278 407 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 394(G) −290 −847 −1769 −1799 −2291

−1705 −1729 −1796 −2216 −1592 −1322 −1804 −1637 −1895 −526 −654 179 −2597 −2248 408 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 395(A)

−539 −1889 −1592 −1657 −1048 −1298 −901 −1422 −1478 −795 −1130 −1629 −1231 −1567 446 −331 1000 −2050 −1697 409 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −21 −6672 −7714 −894 −1115 −701 −1378 * * 396(R) −1262 −1929 −1576 −1005 −2676 −1901 −641 −2373 404 −2286 −1610 −1018 104 −286

−1282 −1246 −2043 −2243 −1991 410 — −149 −500 233 43 −381 399 106 −626 210 −466 −720 275 394 45 96 359 117 −369 −294 −249 — −30 −6155 −7197 −894 −1115 −701 −1378 * * 397(G) 21 −791 −381 −92 −1740

−286 −1423 81 −1560 −757 −172 −1269 42 694 855 −134 −959 −1829 −1336 411 — * * * * * * * * * * * * * * * * * * * * — * * * * * * * * 0 ¹Program name and version ²Name of the input sequence alignment file ³Length of the alignment: include indels ⁴Type of residues ⁵Map of the match states to the columns of the alignment ⁶Number of sequences in the alignment ⁷When the file was generated ⁸The transition probability distribution for the null model (single G state). ⁹The symbol emission probability distribution for the null model (G tate); consists of K integers. The null probability used to convert these back to model probabilities is 1/K.

TABLE 2 E-values and XI Group membership for proteins by SEQ ID NO. SEQ ID NO: Amino XI Organism acid GI or AC # Group E-value Streptomyces 16 126348424 I 0 ambofaciens Streptomyces sp. 20 38141596 I 0 Streptomyces 32 Q9L0B8 I 0 coelicolor Streptomyces 34 Q93HF3 I 0 avermitilis Streptomyces 68 P09033 I 0 violaceusniger Streptomyces 96 Q93RJ9 I 0 olivaceoviridis Streptomyces 106 Q9S3Z4 I 0 corchorusii Streptomyces 108 Q9L558 I 0 thermocyaneoviolaceus Streptomyces 122 P50910 I 0 diastaticus Streptomyces 128 P24300 I 0 rubiginosus Streptomyces murinus 130 P37031 I 0 Streptomyces 135 P15587 I 0 olivochromogenes Streptomyces 136 157879319 I 0 olivochromogenes Streptomyces 138 157881044 I 0 olivochromogenes Streptomyces 139 7766813 I 0 diastaticus Streptomyces albus 142 P24299 I 0 Streptomyces 144 9256915 I 0 diastaticus Streptomyces 146 21730246 I 0 rubiginosus Streptomyces 48 197776540 I 1.00E−305 pristinaespiralis Actinoplanes 66 P12851 I 2.70E−305 missouriensis Actinoplanes 140 349936 I 1.30E−302 missouriensis Streptomyces sp. 46 197764953 I 2.90E−302 Clavibacter 42 A5CPC1 I 4.30E−302 michiganensis Actinoplanes 134 443486 I 7.80E−302 missouriensis Actinoplanes 147 443568 I 7.80E−302 missouriensis Clavibacter 2 B0RIF1 I 1.40E−301 michiganensis Actinoplanes sp. 24 P10654 I 2.10E−301 Arthrobacter sp. 126 A0JXN9 I 2.30E−301 Actinoplanes 145 443526 I 2.30E−301 missouriensis Actinoplanes 143 443303 I 4.00E−301 missouriensis Arthrobacter sp. 54 P12070 I 3.50E−298 Arthrobacter aurescens 90 119964059 I 1.10E−297 Micromonospora sp. 112 237882534 I 2.00E−297 Arthrobacter 4 220912923 I 3.30E−296 chlorophenolicus Arthrobacter sp. 133 231103 I 3.20E−294 Arthrobacter sp. 141 2914276 I 1.10E−293 Arthrobacter sp. 12 60615686 I 8.70E−293 Acidothermus 30 117929271 I 3.50E−292 cellulolyticus Streptomyces griseus 118 182434863 I 1.50E−291 Salinispora arenicola 18 159039501 I 8.10E−289 Geodermatophilus 64 227404617 I 1.80E−286 obscures Catenulispora acidiphila 104 229246901 I 5.60E−286 Salinispora tropica 44 145596104 I 1.80E−282 marine actinobacterium 110 88856315 I 8.10E−282 Mycobacterium 10 118469437 I 5.30E−281 smegmatis Saccharomonospora 76 229886404 I 3.40E−278 viridis Streptomyces rochei 137 P22857 I 3.40E−275 Mycobacterium 120 120406242 I 7.00E−274 vanbaalenii Streptomyces lividans 78 Q9RFM4 I 1.70E−272 Thermus thermophilus 100 P26997 I 4.80E−272 Nakamurella multipartita 38 229221673 I 1.00E−271 Thermos caldophilus 131 4930285 I 1.90E−269 Thermus caldophilus 132 P56681 I 2.70E−269 Streptosporangium 82 229851079 I 1.90E−264 roseum Beutenbergia cavernae 62 229821786 I 9.10E−262 Cellulomonas flavigena 74 229243977 I 1.10E−261 Kribbella flavida 8 227382478 I 4.80E−261 Thermobifida fusca 114 72162004 I 5.90E−261 Janibacter sp. 58 84495191 I 1.40E−259 Nocardiopsis dassonvillei 36 229207664 I 1.80E−258 Actinosynnema mirum 6 226865307 I 4.70E−257 Nocardioides sp. 84 119716602 I 6.40E−256 Actinomyces urogenitalis 14 227497116 I 3.70E−250 Meiothermus ruber 52 227992647 I 1.40E−245 Brachybacterium faecium 60 237671435 I 3.10E−245 Jonesia denitrificans 94 227383768 I 1.60E−244 Kribbella flavida 86 227381155 I 3.80E−244 Frankia sp. 80 158316430 I 2.50E−242 Actinomyces 70 154508186 I 8.80E−239 odontolyticus Meiothermus silvanus 22 227989553 I 1.90E−229 Leifsonia xyli 92 50954171 I 2.50E−226 Mobiluncus curtisii 26 227493823 I 2.50E−225 Mobiluncus mulieris 72 227875705 I 9.70E−225 Deinococcus 124 94972159 I 1.80E−224 geothermalis Stackebrandtia 98 229862570 I 9.40E−224 nassauensis Roseiflexus sp. 50 148656997 I 5.10E−223 Thermobaculum terrenum 56 227374836 I 5.60E−222 Roseiflexus castenholzii 88 156742580 I 3.10E−221 Herpetosiphon 28 159898286 I 1.10E−204 aurantiacus Xylanimonas 40 227427650 I 1.90E−204 cellulosilytica Herpetosiphon 116 159897776 I 8.70E−203 aurantiacus Acidobacteria 102 94967932 I 2.20E−181 bacterium Geobacillus 305 Q5KYS6 II 1.50E−07 kaustophilus Geobacillus 195 A4IP67 II 3.80E−06 thermodenitrificans Tetragenococcus 180 O82845 II 0.00028 halophilus Bacillus licheniformis 237 P77832 II 0.00036 Mesorhizobium sp. 250 Q11EH9 II 0.00085 Bacillus subtilis 220 P04788 II 0.0038 Thermotoga maritima 207 Q9X1Z5 II 0.0049 Thermotoga sp. 227 B1LB08 II 0.0049 Bacillus 212 A7Z522 II 0.012 amyloliquefaciens Thermotoga petrophila 268 A5ILR5 II 0.03 Bacillus sp. 260 P54272 II 0.041 Bifidobacterium longum 175 B3DR33 II 0.068 Geobacillus 238 P54273 II 0.072 stearothermophilus Bacillus megaterium 214 O08325 II 0.28 Bacillus cereus 228 Q739D2 II 0.46 Thermotoga 232 P45687 II 1.1 neapolitana Fervidobacterium 246 Q6T6K9 II 1.5 gondwanense Bacillus halodurans 277 Q9K993 II 1.7 Lactobacillus pentosus 269 P21938 II 1.8 Staphylococcus xylosus 206 P27157 II 2.5 Bifidobacterium 205 A1A0H0 II 3.9 adolescentis Bacillus clausii 278 Q5WKJ3 II 4.2 Bifidobacterium longum 161 Q8G3Q1 II 11 Lactococcus lactis 270 Q9CFG7 II 15 Bacillus pumilus 296 A8FE33 II 21 Thermoanaerobacterium 284 P30435 II 62 saccharolyticum Thermoanaerobacterium 267 P19148 II 75 thermosulfurigenes Thermoanaerobacter 176 P22842 II 1.10E+02 pseudethanolicus Thermoanaerobacter sp. 172 B0K1L3 II 1.40E+02 Rhizobium 240 Q1MBL8 II 2.40E+02 leguminosarum Thermoanaerobacterium 257 P29441 II 2.90E+02 thermosaccharolyticum Lactobacillus brevis 263 P29443 II 3.00E+02 Lactobacillus brevis 274 Q03TX3 II 3.20E+02 Listeria welshimeri 303 A0AF79 II 3.50E+02 Bacteroides 196 Q8A9M2 II 3.90E+02 thetaiotaomicron Thermoanaerobacter 304 Q9KGU2 II 4.10E+02 yonseiensis Rhizobium etli 290 Q2K433 II 4.20E+02 Salmonella enterica 148 B4T952 II 4.30E+02 Klebsiella pneumoniae 149 P29442 II 4.30E+02 Sinorhizobium meliloti 150 Q92LW9 II 4.30E+02 Escherichia coli 151 Q7A9X4 II 4.30E+02 Salmonella enterica 152 Q5PLM6 II 4.30E+02 Xanthomonas 153 Q3BMF2 II 4.30E+02 campestris Pectobacterium 154 Q6DB05 II 4.30E+02 atrosepticum Rhodopirellula baltica 155 Q7UVG2 II 4.30E+02 Xanthomonas axonopodis 156 Q8PEW5 II 4.30E+02 Xanthomonas oryzae 157 Q5GUF2 II 4.30E+02 Pediococcus pentosaceus 158 Q03HN1 II 4.30E+02 Brucella suis 159 Q8G204 II 4.30E+02 Escherichia coli 160 Q0TBN7 II 4.30E+02 Brucella canis 162 A9M9H3 II 4.30E+02 Burkholderia multivorans 163 A9ARG7 II 4.30E+02 Brucella ovis 164 A5VPA1 II 4.30E+02 Rhizobium etli 165 B3Q0R5 II 4.30E+02 Burkholderia xenovorans 166 Q13RB8 II 4.30E+02 Actinobacillus 167 A3N3K2 II 4.30E+02 pleuropneumoniae Burkholderia cenocepacia 168 B4ENA5 II 4.30E+02 Solibacter usilatus 169 Q022S9 II 4.30E+02 Brucella abortus 170 B2SA37 II 4.30E+02 Rhodobacter sphaeroides 171 A4WVT8 II 4.30E+02 Yersinia 173 Q1C0D3 II 4.30E+02 pseudotuberculosis Xanthomonas oryzae 174 Q5GYQ7 II 4.30E+02 Photobacterium 177 Q6LUY7 II 4.30E+02 profundum Escherichia coli 178 B1LJC7 II 4.30E+02 Agrobacterium 179 Q8U7G6 II 4.30E+02 tumefaciens Salmonella enterica 181 B4TZ55 II 4.30E+02 Yersinia 182 Q8Z9Z1 II 4.30E+02 pseudotuberculosis Yersinia 183 Q1CDB8 II 4.30E+02 pseudotuberculosis Rhodobacter 184 A3PNM4 II 4.30E+02 sphaeroides Brucella abortus 185 Q2YMQ2 II 4.30E+02 Salmonella enterica 186 Q8ZL90 II 4.30E+02 Bacteroides vulgatus 187 A6L792 II 4.30E+02 Xanthomonas 188 Q8PLL9 II 4.30E+02 axonopodis Salmonella enterica 189 Q57IG0 II 4.30E+02 Escherichia coli 190 B7M3I8 II 4.30E+02 Roseobacter denitrificans 191 Q162B6 II 4.30E+02 Bacteroides fragilis 192 Q64U20 II 4.30E+02 Enterobacter sakazakii 193 A7MNI5 II 4.30E+02 Brucella abortus 194 Q57EI4 II 4.30E+02 Haemophilus influenzae 197 A5UCZ3 II 4.30E+02 Yersinia 198 B2K7D2 II 4.30E+02 pseudotuberculosis Xanthomonas campestris 199 Q4UTU6 II 4.30E+02 Haemophilus somnus 200 B0UT19 II 4.30E+02 Pseudoalteromonas 201 Q15PG0 II 4.30E+02 atlantica Escherichia fergusonii 202 B7LTH9 II 4.30E+02 Silicibacter sp. 203 Q1GKQ4 II 4.30E+02 Salmonella enterica 204 B5R4P8 II 4.30E+02 Salmonella enterica 208 A9MUV0 II 4.30E+02 Pseudomonas syringae 209 Q48J73 II 4.30E+02 Shigella boydii 210 Q31V53 II 4.30E+02 Burkholderia ambifaria 211 Q0B1U7 II 4.30E+02 Haemophilus influenzae 213 A5UIN7 II 4.30E+02 Arabidopsis thaliana 215 Q9FKK7 II 4.30E+02 Escherichia coli 216 Q3YVV0 II 4.30E+02 Bacteroides fragilis 217 Q5LCV9 II 4.30E+02 Pseudomonas fluorescens 218 Q3KDW0 II 4.30E+02 Escherichia coli 219 B1X8I1 II 4.30E+02 Xanthomonas campestris 221 Q4UNZ4 II 4.30E+02 Pseudomonas syringae 222 Q4ZSF5 II 4.30E+02 Sinorhizobium medicae 223 A6UD89 II 4.30E+02 Ochrobactrum anthropi 224 A6X4G3 II 4.30E+02 Burkholderia 225 Q2SW40 II 4.30E+02 thailandensis Salmonella enterica 226 B5EX72 II 4.30E+02 Salmonella enterica 229 B4SWK9 II 4.30E+02 Salmonella enterica 230 Q7C637 II 4.30E+02 Enterococcus faecalis 231 Q7C3R3 II 4.30E+02 Escherichia coli 233 B7MES1 II 4.30E+02 Photorhabdus 234 Q7N4P7 II 4.30E+02 luminescens Enterobacter sp. 235 A4W566 II 4.30E+02 Burkholderia 236 B1KB47 II 4.30E+02 cenocepacia Brucella abortus 239 Q8YFX5 II 4.30E+02 Yersinia enterocolitica 241 A1JT10 II 4.30E+02 Serratia 242 A8G7W8 II 4.30E+02 proteamaculans Yersinia 243 A7FP68 II 4.30E+02 pseudotuberculosis Escherichia coli 244 B7NEL7 II 4.30E+02 Yersinia pestis 245 A9R5Q1 II 4.30E+02 Xanthomonas 247 Q8P3H1 II 4.30E+02 campestris Rhizobium 248 B5ZQV6 II 4.30E+02 leguminosarum Bradyrhizobium 249 Q89VC7 II 4.30E+02 japonicum Actinobacillus 251 B3H2X9 II 4.30E+02 pleuropneumoniae Yersinia 252 Q663Y3 II 4.30E+02 pseudotuberculosis Xanthomonas 253 Q8P9T9 II 4.30E+02 campestris Burkholderia 254 A0KE56 II 4.30E+02 cenocepacia Oceanobacillus 255 Q8ELU7 II 4.30E+02 iheyensis Brucella suis 256 B0CKM9 II 4.30E+02 Burkholderia 258 B2JFE9 II 4.30E+02 phymatum Yersinia 259 B1JH40 II 4.30E+02 pseudotuberculosis Lactococcus lactis 261 Q02Y75 II 4.30E+02 Novosphingobium 262 Q2GAB9 II 4.30E+02 aromaticivorans Mesorhizobium loti 264 Q98CR8 II 4.30E+02 Escherichia coli 265 A8A623 II 4.30E+02 Burkholderia 266 Q1BG90 II 4.30E+02 cenocepacia Ruminococcus 271 Q9S306 II 4.30E+02 flavefaciens Burkholderia 272 B2T929 II 4.30E+02 phytofirmans Salmonella enterica 273 B5FLD6 II 4.30E+02 Burkholderia ambifaria 275 B1Z405 II 4.30E+02 Salmonella enterica 276 B5RGL6 II 4.30E+02 Marinomonas sp. 279 A6VWH1 II 4.30E+02 Yersinia 280 A4TS63 II 4.30E+02 pseudotuberculosis Actinobacillus 281 B0BTI9 II 4.30E+02 pleuropneumoniae Silicibacter pomeroyi 282 Q5LV46 II 4.30E+02 Xanthomonas oryzae 283 Q2NXR2 II 4.30E+02 Escherichia coli 285 B6I3D6 II 4.30E+02 Escherichia coli 286 B5YVL8 II 4.30E+02 Escherichia coli 287 B7NP65 II 4.30E+02 Escherichia coli 288 B2U560 II 4.30E+02 Escherichia coli 289 B1IZM7 II 4.30E+02 Escherichia coli 291 P00944 II 4.30E+02 Hordeum vulgare 292 Q40082 II 4.30E+02 Dinoroseobacter 293 A8LP53 II 4.30E+02 shibae Rhodobacter 294 Q3IYM4 II 4.30E+02 sphaeroides Actinobacillus 295 A6VLM8 II 4.30E+02 succinogenes Escherichia coli 297 Q8FCE3 II 4.30E+02 Pseudomonas syringae 298 Q880Z4 II 4.30E+02 Burkholderia 299 A4JSU5 II 4.30E+02 vietnamiensis Escherichia coli 300 A7ZTB2 II 4.30E+02 Haemophilus influenzae 301 P44398 II 4.30E+02 Haemophilus influenzae 302 Q4QLI2 II 4.30E+02 Mannheimia 306 Q65PY0 II 4.30E+02 succiniciproducens 

What is claimed is:
 1. A recombinant bacterial strain selected from the group consisting of Zymomonas and Zymobacter comprising a heterologous nucleic acid molecule encoding a polypeptide having xylose isomerase activity wherein the polypeptide is a Group I xylose isomerase and is included in the class of enzymes identified by EC 5.3.1.5, and wherein the strain utilizes xylose as a carbon source and wherein the heterologous nucleic acid molecule encoding a polypeptide having xylose isomerase activity is not under the control of a promoter derived from Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase gene that has a base substitution in a position selected from the group consisting of position 116, position 217, and both position 116 and 217; wherein the position numbers are of SEQ ID NO:311; and wherein the strain demonstrates an increase in xylose utilization of between about 1.9 fold to about 17.1 fold as compared with a strain expressing a Group II xylose isomerase.
 2. A recombinant bacterial strain of claim 1 wherein the polypeptide having xylose isomerase activity gives an E-value score of 1E-15 or less when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, and 142; the query being carried out using the hmmsearch algorithm wherein the Z parameter is set to 1 billion.
 3. A recombinant bacterial strain of claim 1 wherein the polypeptide having xylose isomerase activity: 1) has the following conserved amino acids when compared with the reference amino acid sequence of SEQ ID NO:66: a) leucine at position 226, b) methionine at position 223, c) isoleucine at position 191, d) threonine, serine, or valine at position 195, e) methionine, threonine or guanine at position 88, f) histidine at position 290, g) glutamic acid or aspartic acid at position 221, h) phenylalanine, valine, or leucine at position 242, i) histidine at position 243, j) leucine, phenylalanine, or methionine at position 193, k) glutamine at position 256, l) glycine at position 213, m) proline, tyrosine, alanine, or serine at position 288, and n) glutamine at position 249; or 2) has at least 90% of the conserved amino acids of part (1).
 4. A recombinant bacterial strain of claim 1 wherein the xylose isomerase has an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
 5. A recombinant bacterial strain of claim 1 wherein the xylose isomerase is isolated from a microorganism selected from the group consisting of Actinoplanes, Arthrobacter, Streptomyces, Thermus, Thermobaculum, Herpetosiphon, Acidobacteria, Roseiflexus, Meiothermus, Deinococcus, Meiothermus, Stackebrandtia, Kribbella, Xylanimonas, Nocardiopsis, Catenulispora, Streptosporangium, Geodermatophilus, Actinosynnema, Saccharomonospora, Acidothermus, Tthermobifida, Nocardiodes, Janibacter, Mycobacterium, Leifsonia, Clavibacter, Micromonospora, Salinispora, Cellulomonas, Jonesia, Nakamurella, Actinomyces, Mobiluncus, Brachybacterium, Beutengergai, Frankia, and Actinobacterium.
 6. A recombinant bacterial strain of claim 5 wherein the xylose isomerase is isolated from Actinoplanes missouriensis.
 7. A recombinant bacterial strain of claim 4 wherein the Group I polypeptide having xylose isomerase activity has at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:24, 66, 134, 140, 143, 145, and 147, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
 8. A method for the production of ethanol comprising: a) providing the recombinant bacterial strain of claim 1; and b) contacting the strain of (a) with xylose under conditions whereby the strain produces ethanol.
 9. A method according to claim 8 wherein the polypeptide having xylose isomerase activity gives an E-value score of 1E-15 or less when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs: 2, 24, 32, 34, 42, 54, 66, 68, 78, 96, 100, 106, 108, 122, 126, 128, 130, 132, 135, 137, and 142; the query being carried out using the hmmsearch algorithm wherein the Z parameter is set to 1 billion.
 10. A method according to claim 8 wherein the polypeptide having xylose isomerase activity: 1) has the following conserved amino acids when compared with the reference amino acid sequence of SEQ ID NO:66: a) leucine at position 226, b) methionine at position 223, c) isoleucine at position 191, d) threonine, serine, or valine at position 195, e) methionine, threonine or guanine at position 88, f) histidine at position 290, g) glutamic acid or aspartic acid at position 221, h) phenylalanine, valine, or leucine at position 242, i) histidine at position 243, j) leucine, phenylalanine, or methionine at position 193, k) glutamine at position 256, l) glycine at position 213, m) proline, tyrosine, alanine, or serine at position 288, and n) glutamine at position 249; or 2) has at least 90% of the conserved amino acids of part (1).
 11. A method according to claim 8 wherein the xylose isomerase has an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 2, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, and 147 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
 12. A method according to claim 8 wherein the xylose isomerase is isolated from a microorganism selected from the group consisting of Actinoplanes, Arthrobacter, Streptomyces, Thermus, Thermobaculum, Herpetosiphon, Acidobacteria, Roseiflexus, Meiothermus, Deinococcus, Meiothermus, Stackebrandtia, Kribbella, Xylanimonas, Nocardiopsis, Catenulispora, Streptosporangium, Geodermatophilus, Actinosynnema, Saccharomonospora, Acidothermus, Tthermobifida, Nocardiodes, janibacter, Mycobacterium, Leifsonia, Clavibacter, Micromonospora, Salinispora, Cellulomonas, jonesia, Nakamurella, Actinomyces, Mobiluncus, Brachybacterium, Beutengergai, Frankia, and Actinobacterium. 